Modified yeast strains exhibiting enhanced fermentation of lignocellulosic hydrolysates

ABSTRACT

The present invention relates to novel xylose-fermenting yeast strains (for example, yeast of the genus  Saccharomyces , e.g.,  S. cerevisiae ) with an enhanced ability to ferment the xylose (and/or another pentose sugar) present in a lignocellulosic hydrolysate to a fermentation product(s) (for example, an alcohol (e.g., ethanol) or a sugar alcohol (e.g., xylitol)).

RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalPatent Application No. 61/291,011, filed Dec. 30, 2009, and No.61/307,536, filed Feb. 24, 2010; the content of each application ishereby incorporated by reference herein in its entirety.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing filed on Dec. 30, 2010, created/modified on Dec.30, 2010, named SEQLIST01039019800ST25.txt, having a size in bytes of 4kB, is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel yeast strains with enhancedability to utilize the lignocellulosic hydrolysates for growth and/orfermentation.

BACKGROUND

Plant cell walls consist mainly of the large biopolymers cellulose,hemicellulose, lignin, and pectin. Cellulose and hemicelluloseconstitute an important renewable and inexpensive carbon source for theproduction of fermentable sugars. Cellulose consists of D-glucose unitslinked together in linear chains via beta-1,4 glycosidic bonds.Hemicellulose consists primarily of a linear xylan backbone comprisingD-xylose units linked together via beta-1,4 glycosidic bonds andnumerous side chains linked to the xylose units via glycosidic or esterbonds (e.g., L-arabinose, acetic acid, ferulic acid, etc.).

The term lignocellulose is commonly used to describe plant-derivedbiomass comprising cellulose, hemicellulose and lignin. Much attentionand effort has been applied in recent years to the production of fuelsand chemicals, primarily ethanol, from lignocellulose-containingfeedstocks, such as agricultural wastes and forestry wastes, due totheir low cost and wide availability. These agricultural and forestrywastes are typically burned or landfilled; thus, using theselignocellulosic feedstocks for ethanol production offers an attractivealternative to disposal. Yet another advantage of these feedstocks isthat the lignin byproduct, which remains after the cellulose conversionprocess, can be used as a fuel to power the process instead of fossilfuels. Several studies have concluded that, when the entire productionand consumption cycle is taken into account, the use of ethanol producedfrom cellulose generates close to zero greenhouse gases.

In comparison, fuel ethanol from feedstocks such as cornstarch, sugarcane, and sugar beets suffers from the limitation that these feedstocksare already in use as a food source for humans and animals. A furtherdisadvantage of the use of these feedstocks is that fossil fuels areused in the conversion processes. Thus, these processes have only alimited impact on reducing greenhouse gases.

Lignocellulosic biomass has also been considered for producing otherfermentation products besides ethanol. Examples of such products includelactic acid, sorbitol, acetic acid, citric acid, ascorbic acid,propanediol, butanediol, xylitol, acetone, and butanol.

The first chemical processing step for converting lignocellulosicfeedstock to ethanol or other fermentation products involves hydrolysisof the cellulose and hemicellulose polymers to sugar monomers, such asglucose and xylose, which can be converted to ethanol or otherfermentation products in a subsequent fermentation step. Hydrolysis ofthe cellulose and hemicellulose can be achieved with a single-stepchemical treatment or with a two-step process with milder chemicalpretreatment followed by enzymatic hydrolysis of the pretreatedlignocellulose with cellulase enzymes.

In a single-step chemical treatment, the lignocellulosic feedstock iscontacted with a strong acid or alkali under conditions sufficient tohydrolyze both the cellulose and hemicellulose components of thefeedstock to sugar monomers.

In a two-step chemi-enzymatic hydrolysis process, the lignocellulosicfeedstock is first subjected to a pretreatment under conditions that aresimilar to, but milder than, those in the concentrated acid or alkalihydrolysis process. The purpose of the pretreatment is to increase thecellulose surface area and convert the fibrous feedstock to a muddytexture, with limited conversion of the cellulose to glucose. If thepretreatment is conducted with acid, the hemicellulose component of thefeedstock is hydrolyzed to xylose, arabinose, galactose, and mannose.The resulting hydrolysate, which is enriched in pentose sugars derivedfrom the hemicellulose, may be separated from the solids and used in asubsequent fermentation process to convert the pentose sugars to ethanolor other products. If the pretreatment is conducted with alkali, verylittle hydrolysis of the polysaccharides occurs; however, the alkalitreatment opens up the surface of the lignocellulose by reacting withacidic groups present on the hemicellulose.

After the pretreatment step, the cellulose is subjected to enzymatichydrolysis with one or more cellulase enzymes such asexo-cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidases(beta-G). The CBH and EG enzymes catalyze the hydrolysis of theβ-1,4-D-glucan linkages in the cellulose. The CBH enzymes, e.g., CBHIand CBHII, act on the ends of the glucose polymers in cellulosemicrofibrils and liberate cellobiose, while the EG enzymes act at randomlocations within the cellulose polymer. Together, the cellulase enzymeshydrolyze cellulose to cellobiose, which, in turn, is hydrolyzed toglucose by beta-G. In addition to the CBH, EG, and beta-G enzymes, otherenzymes or proteins that enhance the enzymatic degradation of thepretreated lignocellulosic substrate may be present during thehydrolysis reaction, e.g., xylanases, beta-xylosidases, beta-mannanase,acetyl xylan esterases, ferulic acid esterases, swollenins, andexpansins. The presence of xylanases may be advantageous, for example,in cases where significant amounts of xylan are present in thepretreated feedstock.

If the pentose sugars are separated from the solids between thepretreatment and enzymatic hydrolysis steps, glucose will be the mainsugar monomer in the hydrolysate produced by the enzymatic treatment. Ifthe pentose sugars released by the chemical pretreatment step arecarried through to the enzymatic hydrolysis step, the hydrolysate willcontain glucose and xylose in about a 2:1 weight ratio, with L-arabinosebeing about 3-5 wt % of the total sugar monomers. Conversion of thehexose and pentose sugars in the resulting lignocellulosic hydrolysate(sometimes referred to as a lignocellulose hydrolysate) to ethanol oranother product(s) is carried out in a subsequent microbialfermentation.

If glucose is the predominant sugar present in the hydrolysate, thefermentation is typically carried out with a Saccharomyces spp. yeast,which converts this sugar and other hexose sugars present into ethanol.However, if the hydrolysate comprises a significant proportion ofpentose sugars, such as xylose and arabinose derived from hemicellulose,the fermentation is carried out with a microbe that naturally possesses,or has been engineered to possess, the ability to ferment xylose and/orarabinose to ethanol or another product(s).

Examples of microbes that can naturally grow on and/or ferment pentosesugars, such as xylose or arabinose, to ethanol or sugar alcoholsinclude, but are not limited to, certain species of yeasts from thegenera Candida, Pichia, and Kluyveromyces. However, such yeaststypically ferment glucose at a much slower rate than Saccharomyces. Thisis a particularly significant limitation in a process for fermentinglignocellulosic hydrolysate containing large proportions of glucose andxylose.

Examples of microbes that have been genetically modified to utilizexylose for growth or fermentation include, but are not limited to,recombinant Saccharomyces strains into which has been inserted either(a) the xylose reductase (XR)-encoding and xylitol dehydrogenase(XDH)-encoding genes (XYL1 and XYL2, respectively) from Pichia stipitis(U.S. Pat. Nos. 5,789,210, 5,866,382, 6,582,944, and 7,527,927, andEuropean Patent No. 450530) or (b) a fungal or bacterial xyloseisomerase (XI) gene (U.S. Pat. Nos. 6,475,768 and 7,622,284). Suchstrains are able to utilize both xylose and glucose for growth and/orfermentation. Examples of yeasts that have been genetically modified toutilize L-arabinose for growth and/or fermentation include, but are notlimited to, recombinant Saccharomyces strains into which genes fromeither fungal (U.S. Pat. No. 7,527,951) or bacterial (InternationalPatent Pub. No. WO 2008/041840) arabinose metabolic pathways have beeninserted. In some instances, recombinant Saccharomyces strains have beendeveloped to utilize both xylose and arabinose for growth and/orfermentation (International Patent Pub. No. WO 2006/096130). Furthergenetic modifications to such strains have been made, by geneticengineering and/or adaptive evolution techniques, to enhance the xyloseconversion rate or ethanol yield from xylose. These modificationsinclude overexpression of sugar transporters (U.S. Patent Pub. No.2007/0082386), deletion of endogenous nonspecific aldose reductase GRE3(U.S. Pat. No. 6,410,302), and enhancement in the pentose phosphatepathway (International Patent Pub. No. WO 2005/108552; U.S. Patent Pub.Nos. 2006/0216804 and 2007/0082386). However, whereas the xyloseconversion rates and/or yields of ethanol from xylose were increasedwith pure sugar fermentations, similar results were either not observedor not reported for fermentations of pentose sugars in lignocellulosichydrolysates.

Lignocellulosic hydrolysates, regardless of whether produced by asingle-step chemical treatment process or by a two-step chemicalpretreatment and enzymatic hydrolysis process, typically comprise notonly sugar monomers such as glucose, xylose and arabinose, but alsolignin monomers, acetate (released from the hemicellulose side-chains),and chemical reaction products of the sugars, such as furfural (fromxylose) and hydroxymethylfurfural (from glucose). Acetate, furfural, andhydroxymethylfurfural are well-known inhibitors of microbial growthand/or fermentation processes converting sugars to ethanol. The presenceof acetic acid in lignocellulosic hydrolysates is especially problematicas it inhibits yeast cell growth and thus can significantly reduce theyield of fermentation products (Abbott et al. (2007) FEMS Yeast Res.7:819-33). Other yeast inhibitors that arise when convertinglignocellulosic feedstocks to fermentable sugars are furfural and5-hydroxymethylfurfural (HMF). Furfural and HMF result from the loss ofwater molecules from xylose and glucose, respectively, by exposure tohigh temperatures and acid. The inhibitory effects of these compoundsdecrease the efficiency of the fermentation operations by lengtheningthe time required for carrying out the fermentation, increasing theamount of yeast required, decreasing the final yields, or a combinationof these.

It is possible to remove these inhibitors from the lignocellulosichydrolysate prior to their conversion to ethanol (or other chemicals) ina yeast fermentation by physical separation methods. However, theseprocesses are often costly and are likely to result in an increase inoverall costs for the production of the ethanol or other desiredfermentation product(s) from the lignocellulosic biomass.

For example, one method that has been proposed to reduce theconcentration of inhibitors arising from hydrolysis of lignocellulosicfeedstocks is overliming, which involves the addition of Ca(OH)₂ toprecipitate inhibitors from lignocellulosic hydrolysates, therebyimproving the subsequent fermentation using yeast. Such processes aredisclosed by U.S. Pat. Nos. 2,203,360; 4,342,831; 6,737,258; 7,455,997;and Wooley et al. (In: Lignocellulosic Biomass to Ethanol Process Designand Economics Utilizing Co-Current Dilute Acid Prehydrolysis and EnzymeHydrolysis Current and Future Scenarios (1999) Technical Report,National Renewable Energy Laboratory, pp. 16-17). However, any handlingof the lime cake is difficult and costly. In addition, the introductionof calcium into the stream increases the likelihood that calcium scalewill deposit on evaporators, distillation columns, and other processequipment. The clean-up and avoidance of scale increases the cost ofsugar processing.

Another method that has been proposed to remove inhibitors offermentation is ion exchange. For example, ion exchange has beeninvestigated by Nilvebrant et al. ((2001) App. Biochem. Biotech.91-93:35-49) in which a spruce hydrolysate was treated to removefermentation inhibitors, such as phenolic compounds, furan aldehydes,and aliphatic acids. U.S. Pat. No. 7,455,997 and Wooley et al. (supra)report the use of ion exchange to remove acetic acid from anacid-hydrolyzed mixture obtained from wood chips, followed by limetreatment. Similarly, Watson et al. ((1984) Enzyme Microb. Technol.6:451-56) disclose the use of ion exchange to remove inhibitors, such asacetic acid and 2-furaldehyde (furfural), from a sugar cane bagasse acidhydrolysate prior to fermentation. Furthermore, Tran and Chambers((1986) Enzyme Microb. Technol. 8:439-44) disclose various treatments toremove inhibitors prior to fermentation from an acid prehydrolysate fromred oak, including mixed bed ion resin treatment.

In practice, several factors limit the effectiveness of ion exchangetreatment to remove inhibitors. First, the multi-component nature of thestreams results in an inefficient removal of some species at any singleset of conditions. Second, the high ionic load demands very frequent andexpensive regeneration of the resin. Finally, not all of the inhibitorsare ionic, and ion exchange is ineffective in removing nonioniccompounds from sugar.

U.S. Patent Pub. No. 2008/0171370 reports that gallic acid can be usedto detoxify hydrolysates resulting from pretreating a lignocellulosicmaterial by binding acetic acid. As disclosed therein, the gallic acidis a natural polymer comonomer, i.e., the core of the gallotanninstructure, and therefore is a natural means to polymerize phenols andacetic acid in a Fischer esterification with a sulfuric acid catalyst.

International Patent Pub. No. WO 2008/124162 discloses the selectiveremoval of acetate from a sugar mixture containing xylose and glucose byan E. coli strain that is able to convert acetate to a biochemical suchas ethanol, butanol, succinate, lactate, fumarate, pyruvate, butyricacid, and acetone. The E. coli has been deleted in four genes that wouldotherwise code for proteins involved in xylose and glucoseutilization—thereby preventing the consumption of either xylose orglucose by the E. coli—but that have no known effect on acetatemetabolism. After acetate conversion to a biochemical, xylose andglucose fermentation are conducted on the sugar mixture using separatemicroorganisms, one with the ability only to ferment xylose, and theother with the ability only to ferment glucose. However, the process isnot directed to removing unwanted sugars from a sugar hydrolysate, butrather to maximizing the conversion of all sugars present in the mixtureto ethanol or other biochemicals.

An alternative approach to detoxification of the lignocellulosichydrolysate is to develop yeast strains that are tolerant of theinhibitory compounds present in such hydrolysates. For example,adaptation to lignocellulose hydrolysates has been reported for Pichiaand Saccharomyces strains (Huang et al. (2009) Bioresource Technol.100:3914-20; Martin et al. (2007) Bioresource Technol. 98:1767-73).Although this approach generated strains with some tolerance to thehydrolysate, the genotype(s) of the adapted strains was notcharacterized.

Other attempts to characterize and/or improve tolerance tolignocellulose hydrolysates have been directed towards tolerance to thethree major inhibitors in lignocellulosic hydrolysates—acetic acid,furfural, and hydroxymethylfurfural (HMF). For example, strains ofPichia and Saccharomyces have been adapted to media containing furfuraland/or hydroxymethylfurfural (Liu et al. (2004) J. Ind. Microbiol.Biotechnol. 31:345-52; Liu et al. (2005) Appl. Biochem. Biotechnol.121-124:451-60). Other studies have attempted to identify genes thatcontribute to inhibitor tolerance by screening collections of yeastsingle-deletion strains to identify those deletions that increase thesensitivity of the yeast to furfural (Gorsich et al. (2006) Appl.Microbiol. Biotechnol. 71:339-49), acetaldehyde (Matsufuhi et al. (2008)Yeast 25:825-22), and lactic and acetic acids (Kawahata et al. (2006)FEMS Yeast Research 6:924-36). Expression profiling of a limited set ofgenes was conducted to determine changes in gene expression in a yeaststrain adapted to furfural and HMF vs. a parental strain cultured in thepresence of HMF (Liu et al. (2009) Mol. Genet. Genomics 282:233-44).However, neither the sensitivity of the resulting strains to, nor theexpression profile of the strains cultured in the presence of,lignocellulose hydrolysates was reported.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide yeast strains withenhanced ability to utilize the xylose present in a lignocellulosichydrolysate for growth and/or fermentation to ethanol or otherfermentation product(s).

The present invention provides a modified yeast strain capable ofutilizing xylose and/or other pentose sugars present in alignocellulosic hydrolysate for growth and/or fermentation of suchpentose sugars to a fermentation product (e.g., an alcohol (e.g.,ethanol) or a sugar alcohol (e.g., xylitol)). The modified yeast strainexhibits an increase in growth rate of at least 1.3-fold (e.g.,1.5-fold, 2-fold, or greater) in the specific rate of xylosefermentation relative to a corresponding parental yeast strain fromwhich the modified yeast strain is derived. The isolated, modified yeaststrain may be Saccharomyces, or may be some other genus of yeast.

In an aspect of the invention, the modified strain comprises, relativeto a parental strain from which it is derived, (a) an increase in copynumber or expression (e.g., copy number and/or expression) of one ormore genes selected from the group consisting of ARE1, VBA3, RNQ1, FUS1,PDI1, BUD23, IMG1, DDI2, SNO3, SNZ3, YCL042W, YCL073C, YCR045C, andtE(UUC)B (and in some additional embodiments, BIK1 and GLK1), and/or (b)a decrease in copy number or expression (e.g., copy number and/orexpression) of one or more genes selected from the group consisting ofALD2, SSA2, MST27, PRM8, ERV14, ECM21, ILS1, PRP6, YBL100W-C, YBR201C-A,and tE(UUC)G2 (and in some additional embodiments, ALD3).

In another aspect of the invention, the modified strain comprises,relative to a parental strain from which it is derived, (a) an increasein copy number and/or expression of one or more genes of the galactosemetabolic pathway selected from the group consisting of GAL1, GAL7,GAL10, GAL80, and PGM1, and/or (b) a decrease in the copy number and/orexpression of one or more genes of the galactose metabolic pathwayselected from the group consisting of GAL3, GAL4, GAL11, and PGM2.

In yet another aspect of the invention, the modified strain comprises,relative to a parental strain from which it is derived, (a) an increasein copy number and/or expression of one or more genes involved in hexosetransport selected from the group consisting of HXT1, HXT2, HXT3, GAL2,HXK2, and GRR1, and/or (b) a decrease in copy number and/or expressionof one or more genes involved in hexose transport selected from thegroup consisting of HXT4, HXT5, HXT6, HXT7, HXT9, HXT11, HXT12, SNF3,RTG2, and REG1.

In still another aspect of the invention, the modified strain comprises,relative to a parental strain from which it is derived, an increase incopy number and/or expression of one or more genes of the ergosterolbiosynthetic pathway selected from the group consisting of ERG1, ERG8,ERG10, ERG10, ERG20, ERG25, ERG26, ERG27, HMG1, and CYB5 (and, in someadditional embodiments, ERG2, ERG3, ERG4, ERG5, ERG24, and ERG28).

For the purposes of the invention described herein, the modified yeaststrain is identical to the parental yeast strain except for (a) anincrease in copy number and/or expression of one or more genes selectedfrom the group consisting of ARE1, VBA3, RNQ1, FUS1, PDI1, BUD23, IMG1,DDI2, SNO3, SNZ3, YCL042W, YCL073C, YCR045C, tE(UUC)B, GAL1, GAL7,GAL10, GAL80, PGM1, HXT1, HXT2, HXT3, GAL2, HXK2, GRR1, ERG1, ERG8,ERG10, ERG11, ERG20, ERG25, ERG26, ERG27, HMG1, and CYB5 (and, in someadditional embodiments, BIK1, GLK1, ERG2, ERG3, ERG4, ERG5, ERG24, andERG28), and/or (b) a decrease in copy number and/or expression of one ormore genes selected from the group consisting of ALD2, SSA2, MST27,PRM8, ERV14, ECM21, ILS1, PRP6, YBL100W-C, YBR201C-A, tE(UUC)G2, GAL3,GAL4, GAL11, PGM2, HXT4, HXT5, HXT6, HXT7, HXT9, HXT11, HXT12, SNF3,RTG2, and REG1 (and in some additional embodiments, ALD3).

The modified yeast strain may be derived from a parental yeast strainthat is naturally capable of glucose fermentation, e.g., a species ofSaccharomyces. In some embodiments, the modified yeast strain is capableof fermenting both the glucose and xylose present in lignocellulosehydrolysates. The modified yeast strain may also be derived from aparental yeast strain that is naturally capable of xylose fermentation,e.g., a species of Candida, Pichia, or Kluyveromyces, or one that hasbeen modified for enhanced xylose utilization through recombinant ornonrecombinant means.

The modified yeast strain may be derived from a parental strain ofSaccharomyces that has been made capable of utilizing xylose for growthor fermentation by incorporation of (a) genes encoding xylose reductase(XR) and xylitol dehydrogenase (XDH) and/or (b) gene(s) encoding one ormore xylose isomerase (XI). In addition, the modified yeast strain mayalso overexpress an endogenous or heterologous gene encodingxylulokinase. Alternatively, the modified yeast strain may be derivedfrom a parental strain of Saccharomyces that has been made capable ofutilizing xylose for growth or fermentation by one or morenonrecombinant methods, such as adaptive evolution or random mutagenesisand selection. The practice of the invention is not limited by the meansused to produce the modified yeast strain and/or the parental yeaststrain.

The modified yeast strain may comprise other modifications that enableenhanced fermentation of xylose or other sugars present inlignocellulose hydrolysates, including but not limited to, alteredexpression of one or more genes encoding enzymes of the pentosephosphate pathway (PPP), decreased expression of one or more genesencoding nonspecific aldose reductase(s), or expression of one or moregene encoding enzymes enabling fermentation of L-arabinose or otherhexose or pentose sugars present in lignocellulose hydrolysates (e.g.,mannose, galactose, and fucose). In some embodiments, these same othermodifications are also present in the parental yeast strain. Thus, theseother modifications may be introduced to the parental yeast strainbefore, during, or after the modifications provided by the presentinvention.

One modified yeast strain of the present invention, S. cerevisiaeY108-1, has been deposited with the American Type Culture Collection(ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209. Thedeposit was made on Jan. 6, 2010, and was assigned ATCC Deposit No.PTA-10567.

The present invention also relates to a process for producing afermentation product (e.g., an alcohol (e.g., ethanol) or a sugaralcohol (e.g., xylitol)) from a lignocellulosic hydrolysate comprisingfrom about 30 wt % to about 90 wt % xylose (as a function of the weightof total carbohydrate) utilizing the modified yeast strain as describedherein. In addition, the present invention contemplates thelignocellulosic hydrolysate containing some percentage(s) of at leastone inhibitor of yeast growth or fermentation (e.g., acetic acid,furfural, HMF) as a function of the total dissolved solids in thehydrolysate. In the fermentation process of the present invention, amodified yeast strain as described above is cultured (e.g., culturedanaerobically) in a fermentation medium comprising the lignocellulosichydrolysate under conditions that facilitate the conversion of thexylose in the hydrolysate to the fermentation product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show comparisons of glucose and xylose consumption andethanol production in lignocellulosic hydrolysate (FIG. 1A) and puresugar media (FIG. 1B). Data presented are averages of experiments (n=3)for each condition and strain. The modified strain (Y108-1) onlyexhibited increased xylose uptake and conversion during the fermentationof lignocellulosic hydrolysate (FIG. 1A), and not in pure sugar media(FIG. 1B), when compared to the parental strain (LNH-ST).

FIG. 2 is a metabolic diagram depicting glycolysis (solid arrows), thepentose phosphate pathway (dashed arrows), and the introduced pathwayrequired for xylose utilization (dotted arrows). Abbreviations: G-6-P(glucose-6-phosphate), F-6-P (fructose-6-phosphate), F-1,6-BP(fructose-1,6-bisphosphate), DHAP (dihydroxyacetone phosphate), GAP(glyceraldehyde-3-phosphate), PYR (pyruvate), Ru-5-P(ribulose-5-phosphate), R-5-P (ribose-5-phosphate), Xu-5-P(xylulose-5-phosphate), S-7-P, (sedoheptulose-7-phosphate), and E-4-P(erythrose-4-phosphate).

FIG. 3 presents a CGH summary plot showing high copy gain in XKS1.

FIG. 4 presents a CGH summary plot showing copy losses in ALD2 and ALD3.

FIG. 5 presents a CGH summary plot showing copy losses in ECM21 andYBL100W-C.

FIG. 6 presents a CGH summary plot showing copy loss in ILS1.

FIG. 7 presents a CGH summary plot showing copy losses in MST27, PRM8,ERV14, and tE(UUC)G2.

FIG. 8 presents the relative transcript levels of the S. cerevisiae XKS1and Pichia stipitis XYL1 and XYL2 genes in modified (Y108-1) andparental (LNH-ST) yeast strains measured using real-time qRT-PCR asdescribed in Example 4.3, and normalized to the transcription levels ofthe RDN18 gene. Error bars represent the standard error of the mean(SEM) for duplicate measurements from each of two biological replicates.

FIG. 9 presents the enzymatic activity of (FIG. 9A) xylose reductase(with NADPH or NADH) and (FIG. 9B) xylitol dehydrogenase and xylulosekinase in parental (LNH-ST, dark grey) and modified (Y108-1, light grey)yeast strains as described in Example 5. Biomass from triplicatefermentations was pooled to produce a single cell-free extract for eachstrain and time point. Enzymatic activities were determined induplicate. Average activities and standard deviations are shown.

FIG. 10 presents the relative transcript levels of the S. cerevisiaeFUS1, YCL042W, YCL073C, and VBA3 genes in modified (Y108-1) and parental(LNH-ST) yeast strains measured by real-time qRT-PCR and normalized tothe transcription levels of the ACT1 gene. Error bars represent thestandard error of the mean (SEM) from each of two biological replicates

FIG. 11 presents the relative transcript ratios (Log₂) of the genesshowing copy number gains or losses in the modified yeast strain(Y108-1) vs. the parental strain (LNH-ST) as assessed by microarrayhybridization performed as described in Example 4.2. Error barsrepresent the standard error of the mean (SEM) for duplicatemeasurements from each of three biological replicates.

FIG. 12 presents the relative transcript ratios (Log₂) of the genesinvolved in galactose metabolism in the modified yeast strain (Y108-1)vs. the parental strain (LNH-ST) as assessed by microarray hybridizationperformed as described in Example 4.2. Error bars represent the standarderror of the mean (SEM) for duplicate measurements from each of threebiological replicates.

FIG. 13 presents the relative transcript ratios (Log₂) of the genesinvolved in hexose transport in the modified yeast strain (Y108-1) vs.the parental strain (LNH-ST) as assessed by microarray hybridizationperformed as described in Example 4.2. Error bars represent the standarderror of the mean (SEM) for duplicate measurements from each of threebiological replicates.

FIG. 14 presents the relative transcript ratios (Log₂) of genes involvedin ergosterol biosynthesis metabolism in the modified yeast strain(Y108-1) vs. the parental strain (LNH-ST) as assessed by microarrayhybridization performed as described in Example 4.2. Error barsrepresent the standard error of the mean (SEM) for duplicatemeasurements from each of three biological replicates.

FIG. 15 presents a map of the plasmid pLNH-ST comprising the P. stipitisXYL1 gene encoding xylose-reductase (XR) linked to the ADH1 promoter(Padh), the P. stipitis XYL2 gene encoding xylitol dehydrogenase (XDH)linked to the pyruvate kinase promoter (Ppyk) and the S. cerevisiae geneencoding xylulose kinase (XKS1) linked to the pyruvate kinase promoter(Ppyk). Other elements include antibiotic selection markers (KanR andAmpR), S. cerevisiae 5S ribosomal DNA targeting sequences (5S rDNA) andan autonomously replicating sequence (ARS).

FIG. 16 presents the comparative growth of a control parental (strainYSC1048) and modified yeast strains overexpressing (+) or deleted in (Δ)the genes shown on (FIG. 16A) synthetic media (SM)+30 g/L xylose platesand (FIG. 16B) media containing dilute, glucose-depleted lignocellulosehydrolysate (10% lignocellulosic hydrolysate). Plates were incubated at30° C. for five days.

FIG. 17 presents the relative growth density on synthetic media+30 g/Lxylose (light grey) or glucose-depleted dilute lignocellulosichydrolysate plates (dark grey) of parental and modified yeast strainscontaining the plasmid pLNH-ST. Plates were imaged and relative densitywas measured with ImageJ software. Dashed lines indicate the averagegrowth density of the modified yeast strains only. Dotted linesillustrate standard deviation of the mean.

FIG. 18 presents the relative transcript level of genes involved in thegalactose metabolic pathway in the modified (Y108-1) vs. the parental(LNH-ST) yeast strain.

FIGS. 19A, 19B, and 19C present the relative transcript level of genesinvolved in the ergosterol biosynthetic pathway in the modified (Y108-1)vs. the parental (LNH-ST) yeast strain.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of an embodiment by way of example only andwithout limitation to the combination of features necessary for carryingthe invention into effect. The headings provided are not meant to belimiting of the various embodiments of the invention. Terms such as“comprises,” “comprising,” “comprise,” “includes,” “including,” and“include” are not meant to be limiting. In addition, the use of thesingular includes the plural, and “or” means “and/or” unless otherwisestated. Unless otherwise defined herein, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art.

Modulation of Genetic Profiles in Modified Yeast Strains

As used herein, a modified yeast strain is a strain that exhibits achange (i.e., an increase or decrease) in copy number or expression ofthe identified gene(s) relative to the copy number or expression of thesame gene(s) in a parental strain from which it is derived.

A parental yeast strain is a strain that is capable of xylosefermentation, either naturally or as the result of mutagenesis, butwhich exhibits wild-type or native copy number or expression of theidentified genes that are increased or decreased in copy number orexpression in the modified yeast strain.

The modified yeast strain may be derived from a parental yeast strain ofSaccharomyces that has been made capable of xylose fermentation byrecombinant incorporation of (a) genes encoding xylose reductase (XR)and xylitol dehydrogenase (XDH) and/or (b) gene(s) encoding one or morexylose isomerase (XI). In addition, the modified yeast strain may alsooverexpress an endogenous or heterologous gene encoding xylulokinase(XK). One such Saccharomyces strain that expresses the XR and XDHencoding genes from Pichia stipitis and overexpresses the endogenous XKencoding gene is Saccharomyces strain LNH-ST (described in U.S. Pat. No.7,527,927).

Alternatively, the modified yeast strain may be derived from a parentalstrain of Saccharomyces that has been made capable of xylosefermentation by one or more nonrecombinant techniques, such as adaptiveevolution or random mutagenesis and selection. However, it should beappreciated that the practice of the invention is not limited by themeans used to produce the parental and modified yeast strains.

The modified yeast strain may also be derived from a parental strain ofCandida, Pichia, or Kluyveromyces that is naturally capable of xylosefermentation, or one that has been modified for enhanced xylosefermentation through recombinant or nonrecombinant means.

For the purposes described herein, the term “increased copy number”means at least one extra copy of at least the coding region of a givengene is present in the modified yeast as compared to the number ofcopies of the same gene in the parental yeast. For example, the modifiedyeast strain may contain 1, 2, 3, 4, 5, 10, or more extra copies of atleast the coding region of a given gene relative to the number of copiesof that same gene in the parental yeast strain. The extra copies of agiven gene may be integrated into the genome of the modified yeaststrain or may be present on one or more autonomously replicating vectorsor plasmids present in the modified yeast strain.

For the purposes described herein, the term “increased expression” meansat least about a 1.3-fold increase in the level of transcript for agiven gene in the modified yeast as compared to the level of transcriptfor the same gene in the parental yeast, when grown under identical ornearly identical conditions of medium composition, temperature, pH, celldensity, and age of culture. For example, the transcript level of agiven gene in the modified yeast can be increased by at least 1.3-,1.5-, 2.0-, 2.5-, 3.0-, 4.0-, 5.0-, or 10-fold, or more, relative to thetranscript level of the same gene in the parental yeast when grown orcultured under essentially the same culture conditions.

For the purposes described herein, the term “decreased copy number”means at least one less copy of at least the coding region of a givengene is present in the genome of the modified yeast as compared to thecopy number of the same gene present in the parental yeast.

For the purposes described herein, the term “decreased expression” meansat least about a 2-fold decrease in the level of transcript for a givengene in the modified yeast as compared to the level of transcript forthe same gene in the parental yeast when grown under identical or nearlyidentical conditions of medium composition, temperature, pH, celldensity, and age of culture. For example, the level of transcript of agiven gene in the modified yeast strain may be decreased by 2-, 2.5-,3-, 5-, 10-, 20-, 50-fold or more relative to the level of transcript ofthat same gene in the parental yeast strain when grown or cultured underessentially the same culture conditions.

In at least some embodiments of the present invention, the increase ordecrease in copy number or expression of the identified gene(s) in amodified yeast strain can be produced by any of various randommutagenesis and selection techniques. For example, the parental yeaststrain may be subjected to irradiation or chemical mutagenesis to createa library of mutated strains, which are then screened for the desiredaltered phenotype; in the present case, that phenotype would include theability to utilize more efficiently the xylose component of alignocellulosic hydrolysate for growth and/or fermentation. Randommutagenesis and selection techniques also include “adaptive evolutiontechniques” or “evolutionary engineering techniques.” As used herein,the term adaptive evolution technique refers to any method or procedureemployed to influence the phenotype and genetic profile of a yeaststrain or organism through the use of exposure to environmentalchallenges, and subsequent selection of the modified and/or improvedyeast strain with the desired altered phenotype and correspondingaltered genetic profile. For example, a parental yeast strain may becultured in media containing initially low, then increasing,concentrations of lignocellulosic hydrolysate to generate a populationof yeast cells with the ability to utilize the xylose component in thehydrolysate for growth or fermentation, from which individual modifiedyeast strains may be isolated, as described in Example 1.

In at least some embodiments of the present invention, the increase ordecrease in copy number or expression of the identified genes in themodified yeast strain can be produced by any of various geneticengineering techniques. As used herein, the term genetic engineeringtechnique refers to any of several well-known techniques for the directmanipulation of an organism's genes. For example, gene knockout(insertion of an inoperative DNA sequence, often replacing theendogenous operative sequence, into an organism's chromosome), geneknock-in (insertion of a protein-coding DNA sequence into an organism'schromosome), and gene knockdown (insertion of DNA sequences that encodeantisense RNA or small interfering RNA, i.e., RNA interference (RNAi))techniques are well known in the art.

Methods for reducing gene expression are well known and can be performedusing any of a variety of methods known in the art. For example, thegene can be modified to disrupt a transcription or translationinitiation sequence or to introduce a frameshift mutation in thetranscript encoding the polypeptide. Other methods of reducing the geneexpression include post-transcriptional RNA silencing methodologies suchas antisense RNA and RNA interference (RNAi). Antisense techniquesinvolve introducing a nucleotide sequence complementary to thetranscript of a target gene such that the complementary antisensenucleotide sequence hybridizes to the target gene transcript, thusreducing or eliminating the number of transcripts available to betranslated into protein. Examples of expressing an antisense RNA areshown in Ngiam et al. (2000) Appl. Environ. Microbiol. 66(2):775-82; andZrenner et al. (1993) Planta. 190(2):247-52, each of which is herebyincorporated by reference herein in its entirety. RNAi methodologiesinclude double stranded RNA (dsRNA), short hairpin RNAs (shRNAs), andsmall interfering RNAs (siRNAs) as known to one of skill in the art, forexample, the techniques of Fire et al. (1998) Nature 391:806-11;Paddison et al. (2002) Genes Dev. 16:948-58; and Miyagishi et al. (2002)Nat. Biotechnol. 20:497-500. The content of each of the above-citedreferences is incorporated by reference herein in its entirety.

Methods for decreasing the expression of a gene also include partial orcomplete deletion of the gene, and disruption or replacement of thepromoter of the gene such that transcription of the gene is greatlyreduced or even inhibited. For example, the promoter of the gene can bereplaced with a weak promoter, as exemplified by U.S. Pat. No.6,933,133, which is incorporated by reference herein in its entirety.Thus, where the weak promoter is operably linked with the codingsequence of an endogenous polypeptide, transcription of that gene willbe greatly reduced or even inhibited.

In some embodiments, the modified yeast strain has been geneticallymodified to at least partially delete one or more of the identifiedgene(s). As used herein, a gene deletion or deletion mutation is amutation in which part of a sequence of the DNA making up the gene ismissing. Thus, a deletion is a loss or replacement of genetic materialresulting in a complete or partial disruption of the sequence of the DNAmaking up the gene. Any number of nucleotides can be deleted, from asingle base to an entire piece of a chromosome. In some embodiments,complete or near-complete deletion of the gene sequence is contemplated.For example, deletion in a gene may be a deletion of at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of thegene.

In the methods of the present invention, the modified yeast strains ofthe invention exhibit increased growth and/or at least a 1.3-fold (e.g.,1.5-fold, 2-fold, or greater) increase in the specific rate of xylosefermentation, relative to a corresponding parental yeast strain fromwhich the modified yeast strain is derived, in lignocellulosichydrolysates.

Sets of Genes Modulated in Modified Yeast Strains

Modified yeast strains of the present invention can exhibit enhancedfermentation and/or growth on pentose sugars, such as xylose, inlignocellulosic hydrolysate. The modified yeast strain may be a strainof Saccharomyces or another yeast genus. In at least one embodiment, themodified strain is derived from a parental S. cerevisiae strain withgenetic modifications inserting the P. stipitis XYL1 (XR) and XYL2 (XDH)genes (e.g., LNH-ST). In another embodiment, the modified yeast strainmay be S. cerevisiae strain Y108-1 (ATCC Deposit No. PTA-10567).

The modifications of the present invention (as produced by the adaptiveevolution of the parent yeast strain) increased the rate of growth on,or fermentation of, xylose in lignocellulosic hydrolysate above the rateseen in the parental strain.

In addition, the modified yeast strains of the present invention mayalso reduce the levels of the inhibitory compounds furfural and HMFpresent in lignocellulosic hydrolysates.

The modified yeast strain (e.g., Y108-1) can comprise increases ordecreases in the copy number or expression of one or more genes, and theenhanced fermentation rates for pentose sugars (e.g., xylose) can be theresult of these modulations, for example, in the levels oftranscription, translation, and activity associated with the genes ortheir encoded proteins.

In the yeast strains studied here, sixteen genes exhibited increases incopy number in the modified yeast strain relative to the parental yeaststrain: ARE1, VBA3, BIK1, RNQ1, FUS1, PDI1, BUD23, IMG1, DDI2, SNO3,SNZ3, YCL042W, YCL073C, YCR045C, tE(UUC)B, and GLK1. Anothertwenty-seven exhibited increased expression in the modified yeast strainrelative to the parental yeast strain when cultured using lignocellulosehydrolysate as a carbon source: HXK2, GRR1, GAL1, GAL7, GAL10, GAL80,PGM1, HXT1, HXT2, HXT3, GAL2, ERG1, ERG2, ERG3, ERG4, ERG5, ERG8, ERG10,ERG11, ERG20, ERG24, ERG25, ERG26, ERG27, ERG28, HMG1, and CYB5. Theincrease in copy number and expression of these 43 genes can be linkedto the enhanced growth or an increase rate of fermentation of the xylosein lignocellulose hydrolysates exhibited by the modified yeast strain,either directly or indirectly (see Table 1 and Examples 2, 3.2, 4.2, and4.3).

Liu et al. ((2009) Mol. Genet. Genomics 282:233-44) disclose differencesin gene expression between lignocellulose hydrolysate-tolerant and-sensitive strains after growth in the presence of HMF and furfural.Among the genes exhibiting higher levels of mRNA in the tolerant strainare GLK1 and ALD2; the former is noted in the present disclosure (above)as elevated in the modified strain (Y108-1), which is capable ofenhanced fermentation of xylose. Interestingly, ALD2 is noted in thepresent disclosure (above) as decreased in copy number in the modifiedstrain; however, Liu et al. were not studying differences in geneexpression related to xylose fermentation. Similarly, Gorsich et al.((2006) Appl. Microbiol. Biotechnol. 71:339-49) disclosed that adeletion of the ERG3 gene reduced the growth of yeast in the presence offurfural, Matsufuji et al. ((2008) Yeast 25:825-33) disclosed that adeletion in the ALD3 gene conferred increased sensitivity toacetaldehyde, and Kawahata et al. ((2006) FEMS Yeast Res. 6:924-36)disclosed that single deletions of the ERG2, ERG3, ERG4, ERG5, ERG6,ERG24, and ERG28 genes led to increased sensitivity of the yeast strainsto organic acids. Interestingly, although Kawahata et al. disclosed thata deletion in the ERV14 gene also increased sensitivity to organicacids, a decrease in ERV14 expression in the modified yeast of thepresent invention resulted in improved fermentation of lignocellulosehydrolysate.

The present invention contemplates the sets of genes that contribute toproducing an enhanced rate of fermentation of a pentose sugar, e.g.,xylose, in lignocellulosic hydrolysate to include one or more of the 43genes identified as having increased copy numbers or expression (whereinBIK1, GLK1, ERG2, ERG3, ERG4, ERG5, ERG24, and ERG28 are included insets containing at least one other of the identified genes). The setscan include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42 or all 43 of the identified genes.

In the yeast strains studied here, twelve genes exhibited decreases incopy number in the modified strain relative to the parental strain:ALD2, SSA2, MST27, PRM8, ERV14, ECM21, ILS1, PRP6, YBL100W-C, YBR201C-A,ALD3, and tE(UUC)G2. Another fourteen genes exhibited decreasedexpression in the modified yeast strain relative to the parental yeaststrain when cultured using lignocellulose hydrolysate as a carbonsource: GAL3, GAL4, GAL11, PGM2, HXT4, HXT5, HXT6, HXT7, HXT9, HXT11,HXT12, SNF3, RTG2, and REG1. The decrease in copy number and expressionof these 26 genes can be linked to the enhanced growth, or an increasedrate of fermentation of the xylose, in lignocellulose hydrolysatesexhibited by the modified yeast strain, either directly or indirectly(see Table 1 and Examples 2, 3.2, 4.2 and 4.3).

The present invention contemplates the sets of genes that contribute toproducing an enhanced rate of fermentation of a pentose sugar, e.g.,xylose, in lignocellulosic hydrolysate to include one or more of thetwenty-six genes identified as having decreased copy numbers orexpression. The sets can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or all 26 of theidentified genes exhibiting decreased copy number or expression.

The present invention further contemplates the sets of genes thatcontribute to producing an enhanced rate of fermentation of a pentosesugar, e.g., xylose, in lignocellulosic hydrolysate to include anycombination of one or more of the 47 genes identified as havingincreased copy numbers or expression and one or more of the 26 genesidentified as having decreased copy numbers or expression. Thesecombined sets can include modulations of from 2 to 73 of the identifiedgenes.

Further information regarding the individual genes whose copy numberand/or expression is increased or decreased in the present invention isknown and readily available to one of ordinary skill in the art. Forexample, wild-type or native sequences of the individual genesexhibiting changes in copy number or expression in the modified yeaststrains of the present invention can be obtained from publicly availablesources including, but not limited to, the Saccharomyces Genome Database(SGD™; located on the web at www.yeastgenome.org); the Pichia stipitiswebpage at the NCBI Entrez Genome Project website (located atwww.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=12845);and the Kluyveromyces lactis webpage at the EMBL-EBI Eukaryotes Genomeswebsite (located at www.ebi.ac.uk/2can/genomes/eukaryotes/Kluyveromyces_lactis.html).

The modulation of copy numbers of genes can be measured by one ofordinary skill in the art through well-known means, for example,comparative genomic hybridization (CGH), Southern blot hybridization, orquantitative real-time PCR (qRT-PCR) from genomic DNA. A CGH method isprovided in Example 3.2.

The modulation of expression of genes also can be measured by one ofordinary skill in the art through analysis of selected mRNA ortranscript levels by well-known means, for example, quantitativereal-time PCR (qRT-PCR) as described in Example 4.3, Northern blothybridization, or global gene expression profiling using cDNA or oligoarray hybridization, as described in Example 4.2.

In at least one modified yeast strain, the parental Saccharomyces strainhas been genetically engineered or modified by insertion of one or morecopies of the Pichia stipitis XYL1 (encoding xylose reductase or “XR”)and XYL2 (encoding xylitol dehydrogenase or “XDH”) into the genome of aSaccharomyces strain. The modified yeast strain of the present inventionexhibits an apparent further increase in the number of copies andexpression of the XR- and XDH-encoding genes. The ADH1 and CDC19promoter sequences associated with the XR and XDH genes (as shown in themap of plasmid pLNH-ST in FIG. 15) also exhibited an increase in copynumber (copy numbers of XR and XDH genes were not directly analyzed asthey are not part of the native S. cerevisiae genome and thereforeprobes for these genes were not present on the commercialoligonucleotide arrays used to determine gene copy number). This isconsistent with the detection of increased transcript levels andenzymatic activity for XR and XDH in the modified yeast strain relativeto the parental yeast strain (Examples 4.3 and 5, FIGS. 8 and 9)

One method of increasing the rate of pentose sugar fermentation (e.g.,xylose fermentation to ethanol) is decreasing the inhibitory effects ofcertain aromatic aldehydes, such as furfural. At least one study(Almeida et al. (2008) Biotechnol. Biofuels 1:12; see also Almeida etal. (2008) Appl. Microbiol. Biotechnol. 78:939-45) disclosed that theNADH cofactor produced by the activity of XDH is required fordetoxification of furfural and HMF. Thus, increased activity of XDH, asis supported by the increased copy number for its promoter, can lead toelevated levels of NADH and subsequent increased detoxification offurfural (Modig et al. (2002) Biochem. J. 363:769-76). Detoxification ofthat inhibitor in lignocellulosic hydrolysate would produce an increasein xylose fermentation.

TABLE 1 Genes Exhibiting Increased or Decreased Copy Number and/orExpression in Modified Yeast Strains Relative to Parental Strain.Systematic Name, Standard Name, and Description are taken from theSaccharomyces Genome Database (see URL: yeastgenome.org). SystematicName Standard Name Description YGR194C XKS1 Xylulokinase, convertsD-xylulose and ATP to xylulose 5- phosphate and ADP; rate limiting stepin fermentation of xylulose; required for xylose fermentation byrecombinant S. cerevisiae strains tE(UUC)B tRNA-Glu; thiolation ofuridine at wobble position (34) requires Ncs6p YCL029C BIK1Microtubule-associated protein, component of the interface betweenmicrotubules and kinetochore, involved in sister chromatid separation;essential in polyploid cells but not in haploid or diploid cells;ortholog of mammalian CLIP-170 YCL028W RNQ1 [PIN(+)] prion, aninfectious protein conformation that is generally an ordered proteinaggregate YCL027W FUS1 Membrane protein localized to the shmoo tip,required for cell fusion; expression regulated by mating pheromone;proposed to coordinate signaling, fusion, and polarization eventsrequired for fusion; potential Cdc28p substrate YCL043C PDI1 Proteindisulfide isomerase, multifunctional protein resident in the endoplasmicreticulum lumen, essential for the formation of disulfide bonds insecretory and cell-surface proteins, unscrambles nonnative disulfidebonds YCL042W Putative protein of unknown function; epitope-taggedprotein localizes to the cytoplasm YCL040W GLK1 Glucokinase, catalyzesthe phosphorylation of glucose at C6 in the first irreversible step ofglucose metabolism; one of three glucose phosphorylating enzymes;expression regulated by nonfermentable carbon sources YCR045C — Putativeprotein of unknown function; nonessential gene identified in a screenfor mutants with decreased levels of rDNA transcription YCR046C IMG1Mitochondrial ribosomal protein of the large subunit, required forrespiration and for maintenance of the mitochondrial genome YCR047CBUD23 Methyltransferase, methylates residue G1575 of 18S rRNA; requiredfor rRNA processing and nuclear export of 40S ribosomal subunitsindependently of methylation activity; diploid mutant displays randombudding pattern YCR048W ARE1 Acyl-CoA:sterol acyltransferase, isozyme ofAre2p; endoplasmic reticulum enzyme that contributes the major sterolesterification activity in the absence of oxygen YCL1073C Protein ofunconfirmed function; displays a topology characteristic of the MajorFacilitators Superfamily of membrane proteins; coding sequence 98%identical to that of YKR106W YCL069W VBA3 Permease of basic amino acidsin the vacuolar membrane YFL061W DDI2 Protein of unknown function;expression is induced over 100-fold by DNA damage; induction decreasedin rad6 and rad18 mutants YFL060C SNO3 Protein of unknown function,nearly identical to Sno2p; expression is induced before the diauxicshift and also in the absence of thiamin YFL059W SNZ3 Member of astationary phase-induced gene family; transcription of SNZ2 is inducedprior to diauxic shift, and also in the absence of thiamin in aThi2p-dependent manner; forms a coregulated gene pair with SNO3 YBL101CECM21 Protein involved in regulating the endocytosis of plasma membraneproteins; identified as a substrate for ubiquitination by Rsp5p anddeubiquitination by Ubp2p; promoter contains several Gcn4p bindingelements YBL076C ILS1 Cytoplasmic isoleucine-tRNA synthetase, target ofthe G1- specific inhibitor reveromycin A YBL100W-C Putative protein ofunknown function YBR055C PRP6 Splicing factor, component of the U4/U6-U5snRNP complex YGL054C ERV14 Protein localized to COPII-coated vesicles,involved in vesicle formation and incorporation of specific secretorycargo; required for the delivery of bud-site selection protein Axl2p tocell surface; related to Drosophila cornichon tE(UUC)G2 tE(UUC)G2tRNA-Glu; thiolation of uridine at wobble position (34) requires Ncs6pYGL053W PRM8 Pheromone-regulated protein with 2 predicted transmembranesegments and an FF sequence, a motif involved in COPII binding; forms acomplex with Prp9p in the ER; member of DUP240 gene family YGL051W MST27Putative integral membrane protein, involved in vesicle formation; formscomplex with Mst28p; member of DUP240 gene family; binds COPI and COPIIvesicles YLL024C SSA2 ATP binding protein involved in protein foldingand vacuolar import of proteins; member of heat shock protein 70 (HSP70)family; associated with the chaperonin- containing T-complex; present inthe cytoplasm, vacuolar membrane and cell wall YMR170C ALD2 Cytoplasmicaldehyde dehydrogenase, involved in ethanol oxidation and beta-alaninebiosynthesis; uses NAD+ as the preferred coenzyme; expression is stressinduced and glucose repressed; very similar to Ald3p YMR169C ALD3Cytoplasmic aldehyde dehydrogenase, involved in beta- alanine synthesis;uses NAD+ as the preferred coenzyme; very similar to Ald2p; expressionis induced by stress and repressed by glucose YBR201C-A Putative proteinof unknown function YGR175C ERG1 Squalene epoxidase, catalyzes theepoxidation of squalene to 2,3-oxidosqualene; plays an essential role inthe ergosterol- biosynthesis pathway and is the specific target of theantifungal drug terbinafine YMR202W ERG2 C-8 sterol isomerase, catalyzesthe isomerization of the delta-8 double bond to the delta-7 position atan intermediate step in ergosterol biosynthesis YLR056W ERG3 C-5 steroldesaturase, catalyzes the introduction of a C-5(6) double bond intoepisterol, a precursor in ergosterol biosynthesis; mutants are viable,but cannot grow on nonfermentable carbon sources YGL012W ERG4 C-24(28)sterol reductase, catalyzes the final step in ergosterol biosynthesis;mutants are viable, but lack ergosterol YMR015C ERG5 C-22 steroldesaturase, a cytochrome P450 enzyme that catalyzes the formation of theC-22(23) double bond in the sterol side chain in ergosterolbiosynthesis; may be a target of azole antifungal drugs YMR220W ERG8Phosphomevalonate kinase, an essential cytosolic enzyme that acts in thebiosynthesis of isoprenoids and sterols, including ergosterol, frommevalonate YPL028W ERG10 Acetyl-CoA C-acetyltransferase (acetoacetyl-CoAthiolase), cytosolic enzyme that transfers an acetyl group from oneacetyl-CoA molecule to another, forming acetoacetyl-CoA; involved in thefirst step in mevalonate biosynthesis YHR007C ERG11 Lanosterol14-alpha-demethylase, catalyzes the C-14 demethylation of lanosterol toform 4,4″-dimethyl cholesta- 8,14,24-triene-3-beta-ol in the ergosterolbiosynthesis pathway; member of the cytochrome P450 family YJL167W ERG20Farnesyl pyrophosphate synthetase, has bothdimethylallyltranstransferase and geranyltranstransferase activities;catalyzes the formation of C15 farnesyl pyrophosphate units forisoprenoid and sterol biosynthesis YNL280C ERG24 C-14 sterol reductase,acts in ergosterol biosynthesis; mutants accumulate the abnormal sterolignosterol (ergosta- 8,14 dienol), and are viable under anaerobic growthconditions but inviable on rich medium under aerobic conditions YGR060WERG25 C-4 methyl sterol oxidase, catalyzes the first of three stepsrequired to remove two C-4 methyl groups from an intermediate inergosterol biosynthesis; mutants accumulate the sterol intermediate4,4-dimethylzymosterol YGL001C ERG26 C-3 sterol dehydrogenase, catalyzesthe second of three steps required to remove two C-4 methyl groups froman intermediate in ergosterol biosynthesis YLR100W ERG27 3-keto sterolreductase, catalyzes the last of three steps required to remove two C-4methyl groups from an intermediate in ergosterol biosynthesis; mutantsare sterol auxotrophs YER044C ERG28 Endoplasmic reticulum membraneprotein, may facilitate protein-protein interactions between the Erg26pdehydrogenase and the Erg27p 3-ketoreductase and/or tether these enzymesto the ER, also interacts with Erg6p YML075C HMG1 One of two isozymes ofHMG-CoA reductase that catalyzes the conversion of HMG-CoA tomevalonate, which is a rate- limiting step in sterol biosynthesis;localizes to the nuclear envelope; overproduction induces the formationof karmellae YBR020W GAL1 Galactokinase, phosphorylatesalpha-D-galactose to alpha- D-galactose-1-phosphate in the first step ofgalactose catabolism; expression regulated by Gal4p YLR081W GAL2Galactose permease, required for utilization of galactose; also able totransport glucose YDR009W GAL3 Transcriptional regulator involved inactivation of the GAL genes in response to galactose; forms a complexwith Gal80p to relieve Gal80p inhibition of Gal4p; binds galactose andATP but does not have galactokinase activity YPL248C GAL4 DNA-bindingtranscription factor required for the activation of the GAL genes inresponse to galactose; repressed by Gal80p and activated by Gal3pYBR018C GAL7 Galactose-1-phosphate uridyl transferase, synthesizesglucose-1-phosphate and UDP-galactose from UDP-D- glucose andalpha-D-galactose-1-phosphate in the second step of galactose catabolismYBR019C GAL10 UDP-glucose-4-epimerase, catalyzes the interconversion ofUDP-galactose and UDP-D-glucose in galactose metabolism; also catalyzesthe conversion of alpha-D- glucose or alpha-D-galactose to theirbeta-anomers YML051W GAL80 Transcriptional regulator involved in therepression of GAL genes in the absence of galactose; inhibitstranscriptional activation by Gal4p; inhibition relieved by Gal3p orGal1p binding YKL127W PGM1 Phosphoglucomutase, minor isoform; catalyzesthe conversion from glucose-1-phosphate to glucose-6- phosphate, whichis a key step in hexose metabolism YMR105C PGM2 Phosphoglucomutase,catalyzes the conversion from glucose-1-phosphate toglucose-6-phosphate, which is a key step in hexose metabolism; functionsas the acceptor for a Glc-phosphotransferase YOL051W GAL11 Subunit ofthe RNA polymerase II mediator complex; associates with core polymerasesubunits to form the RNA polymerase II holoenzyme; affects transcriptionby acting as target of activators and repressors YHR094C HXT1Low-affinity glucose transporter of the major facilitator superfamily,expression is induced by Hxk2p in the presence of glucose and repressedby Rgt1p when glucose is limiting YMR011W HXT2 High-affinity glucosetransporter of the major facilitator superfamily, expression is inducedby low levels of glucose and repressed by high levels of glucose YDR345CHXT3 Low affinity glucose transporter of the major facilitatorsuperfamily, expression is induced in low or high glucose conditionsYHR092C HXT4 High-affinity glucose transporter of the major facilitatorsuperfamily, expression is induced by low levels of glucose andrepressed by high levels of glucose YHR096C HXT5 Hexose transporter withmoderate affinity for glucose, induced in the presence of nonfermentablecarbon sources, induced by a decrease in growth rate, contains anextended N-terminal domain relative to other HXTs YDR343C HXT6High-affinity glucose transporter of the major facilitator superfamily,nearly identical to Hxt7p, expressed at high basal levels relative toother HXTs, repression of expression by high glucose requires SNF3YDR342C HXT7 High-affinity glucose transporter of the major facilitatorsuperfamily, nearly identical to Hxt6p, expressed at high basal levelsrelative to other HXTs, expression repressed by high glucose levelsYJL219W HXT9 Putative hexose transporter that is nearly identical toHxt11p, has similarity to major facilitator superfamily (MFS)transporters, expression of HXT9 is regulated by transcription factorsPdr1p and Pdr3p YOL156W HXT11 Putative hexose transporter that is nearlyidentical to Hxt9p, has similarity to major facilitator superfamily(MFS) transporters and is involved in pleiotropic drug resistanceYIL170W HXT12 Possible pseudogene in strain S288C; YIL170W/HXT12 and theadjacent ORF, YIL171W, together encode a nonfunctional member of thehexose transporter family YGL253W HXK2 Hexokinase isoenzyme 2 thatcatalyzes phosphorylation of glucose in the cytosol; predominanthexokinase during growth on glucose; functions in the nucleus to repressexpression of HXK1 and GLK1 and to induce expression of its own geneYDL194W SNF3 Plasma membrane low glucose sensor that regulates glucosetransport; contains 12 predicted transmembrane segments and a longC-terminal tail required for induction of hexose transporters; alsosenses fructose and mannose; similar to Rgt2p YGL252C RTG2 Sensor ofmitochondrial dysfunction; regulates the subcellular location of Rtg1pand Rtg3p, transcriptional activators of the retrograde (RTG) and TORpathways; Rtg2p is inhibited by the phosphorylated form of Mks1p YDR028CREG1 Regulatory subunit of type 1 protein phosphatase Glc7p, involved innegative regulation of glucose-repressible genes YJR090C GRR1 F-boxprotein component of the SCF ubiquitin-ligase complex; involved incarbon catabolite repression, glucose- dependent divalent cationtransport, high-affinity glucose transport, morphogenesis, and sulfitedetoxification YNL111CP CYB5 Cytochrome b5, involved in the sterol andlipid biosynthesis pathways; acts as an electron donor to support sterolC5-6 desaturationExpression of Genes and Corresponding Proteins

Several genes are identified in the present invention as exhibiting again in copy number or increased expression in the modified yeast(Y108-1) relative to the parental yeast (LNH-ST). Yeast strains can beimproved in relation to growth or fermentation of xylose inlignocellulosic hydrolysate and other advantageous modifications byoverexpression of one or more of these genes. Techniques for producingsuch overexpression are well known in the art and include, withoutlimitation, transcriptional, post-transcriptional, and translationalupregulation. The advantageous effects of increasing the expression oractivity of the proteins corresponding to the genes identified asexhibiting increased copy number or expression are also contemplated inthe present invention.

Several other genes are identified in the present invention asexhibiting a loss in copy number or decreased expression in the modifiedyeast (Y108-1) relative to the parental yeast (LNH-ST). Yeast strainscan be improved in relation to growth or fermentation of xylose inlignocellulosic hydrolysate and other advantageous modifications byunderexpression (e.g., inhibition of expression) of one or more of thesegenes. Techniques for producing such underexpression are well known inthe art and include, without limitation, transcriptional,post-transcriptional, and translational down-regulation. For example,expression of these genes can be downregulated by antisenseoligonucleotides, RNA interference, ribozymes, triplex-formingoligonucleotides, etc. The advantageous effects of decreasing theexpression or activity of the proteins corresponding to the genesidentified as exhibiting reduced copy number or expression are alsocontemplated in the present invention.

Production of a Lignocellulosic Hydrolysate

The lignocellulosic hydrolysate for use in the present invention resultsfrom the hydrolysis of a lignocellulosic feedstock. Representativelignocellulosic feedstocks are (1) agricultural wastes, such as cornstover, corn cobs, wheat straw, barley straw, oat straw, rice straw,canola straw, and soybean stover; (2) grasses, such as switch grass,miscanthus, cord grass, and reed canary grass; (3) forestry wastes, suchas aspen wood and sawdust; and (4) sugar processing residues, such asbagasse and beet pulp. The feedstocks preferably contain highconcentrations of cellulose and hemicellulose that are the source of thesugar in the aqueous stream.

Lignocellulosic feedstocks comprise cellulose in an amount greater thanabout 20%, more preferably greater than about 30%, more preferablygreater than about 40% (wt/wt). For example, the lignocellulosicmaterial may comprise from about 20% to about 50% (wt/wt) cellulose, orany amount therebetween. Hemicellulose may be present at 15% to 30%(wt/wt), or any amount therebetween. Furthermore, the lignocellulosicfeedstock comprises lignin in an amount greater than about 10%, moretypically in an amount greater than about 15% (wt/wt). Thelignocellulosic feedstock may also comprise small amounts of sucrose,fructose, and starch.

Pretreatment of the Feedstock

According to one illustrative example of pretreatment of the feedstock,the lignocellulosic hydrolysate fed to the fermentation is a streamresulting from pretreating the feedstock with acid, i.e., ahemicellulosic hydrolysate. The acid pretreatment is intended to delivera sufficient combination of mechanical and chemical action to disruptthe fiber structure of the lignocellulosic feedstock and increase thesurface area of the feedstock to make it accessible to cellulaseenzymes. Preferably, the acid pretreatment is performed so that nearlycomplete hydrolysis of the hemicellulose and only a small amount ofconversion of cellulose to glucose occurs. The majority of the celluloseis hydrolyzed to glucose in a subsequent step that uses cellulaseenzymes, although a small amount of the cellulose can be hydrolyzed inthe acid pretreatment step as well. Typically, a dilute acid, at aconcentration from about 0.02% (wt/wt) to about 5% (wt/wt), or anyamount therebetween (measured as the percentage weight of pure acid inthe total weight of dry feedstock plus aqueous solution) is used for thepretreatment.

A preferred pretreatment, without intending to be limiting, is steamexplosion described in U.S. Pat. No. 4,416,648 (Foody; which isincorporated herein by reference).

Examples of acids that can be used in the process include those selectedfrom the group consisting of sulfuric acid, sulfurous acid, sulfurdioxide, and combinations thereof. Preferably, the acid is sulfuricacid.

The acid pretreatment is preferably carried out at a maximum temperatureof about 160° C. to about 280° C. The time that the feedstock is held atthis temperature may be about 6 seconds to about 600 seconds. In oneexample, the pH of the pretreatment is about 0.4 to about 3.0, or any pHvalue or range therebetween. For example, the pH of the pretreatment maybe 0.4, 1.0, 1.5, 2.0, 2.5 or 3.0. Preferably, the pretreatment iscarried out to minimize the degradation of xylose and the production offurfural. Preferably, the pretreatment is also designed to minimize thedegradation of pentose and hexose sugars generally.

In at least one example, the chemical used for pretreatment of thelignocellulosic feedstock is alkali. The alkali used in the pretreatmentreacts with acidic groups present on the hemicellulose to open up thesurface of the substrate. With alkali pretreatment, acetate is producedfrom acetyl groups present on the hemicellulose component of thefeedstock, although the amount of acetate present will vary depending onthe severity of the treatment. However, in contrast to acidpretreatment, alkali pretreatment methods may or may not hydrolyze xylanto produce xylose.

Examples of alkali that may be used in the pretreatment include ammonia,ammonium hydroxide, potassium hydroxide, and sodium hydroxide. Thepretreatment may also be conducted with alkali that is insoluble inwater, such as lime and magnesium hydroxide, although soluble alkali ispreferred.

An example of a suitable alkali pretreatment is Ammonia FreezeExplosion, Ammonia Fiber Explosion, or Ammonia Fiber Expansion (“AFEX”process). According to this process, the lignocellulosic feedstock iscontacted with ammonia or ammonium hydroxide in a pressure vessel for asufficient time to enable the ammonia or ammonium hydroxide to alter thecrystal structure of the cellulose fibers. The pressure is then rapidlyreduced, which allows the ammonia to flash or boil and explode thecellulose fiber structure (see U.S. Pat. Nos. 5,171,592, 5,037,663,4,600,590, 6,106,888, 4,356,196, 5,939,544, 6,176,176, 5,037,663 and5,171,592, which are each incorporated herein by reference). The flashedammonia may then be recovered according to known processes.

Another suitable alkali pretreatment for use in the present inventionemploys dilute solutions of ammonia or ammonium hydroxide as set forthin U.S. Publication Nos. 2009/0053770 and 2007/0031918, which are eachincorporated herein by reference.

Yet a further nonlimiting example of a pretreatment process for use inthe present invention includes chemical treatment of the feedstock withorganic solvents. Organic liquids in pretreatment systems are describedby Converse et al. (U.S. Pat. No. 4,556,430; incorporated herein byreference), and such methods have the advantage that the low boilingpoint liquids easily can be recovered and reused. Other pretreatments,such as the Organosolv™ process, also use organic liquids (see U.S. Pat.No. 7,465,791, which is also incorporated herein by reference).Subjecting the feedstock to pressurized water may also be a suitablepretreatment method (see Weil et al. (1997) Appl. Biochem. Biotechnol.68(1-2):21-40, which is incorporated herein by reference).

Processing after Pretreatment

The pretreatment produces a pretreated feedstock composition (e.g., apretreated feedstock slurry) that contains a soluble component includingthe sugars resulting from hydrolysis of the hemicellulose, optionallyacetic acid and other inhibitors, and solids including unhydrolyzedfeedstock and lignin.

According to a further example, the soluble components of the pretreatedfeedstock composition are separated from the solids. The solublefraction, which includes the sugars released during pretreatment andother soluble components, including inhibitors, may then be sent tofermentation. It will be understood, however, that if the hemicelluloseis not effectively hydrolyzed during the pretreatment, it may bedesirable to include a further hydrolysis step or steps with enzymes orby further alkali or acid treatment to produce fermentable sugars.

The foregoing separation may be carried out by washing the pretreatedfeedstock composition with an aqueous solution to produce a wash stream,and a solids stream comprising the unhydrolyzed, pretreated feedstock.Alternatively, the soluble component is separated from the solids bysubjecting the pretreated feedstock composition to a solids-liquidseparation, using known methods such as centrifugation, microfiltration,plate and frame filtration, cross-flow filtration, pressure filtration,vacuum filtration, and the like. Optionally, a washing step may beincorporated into the solids-liquids separation.

The separated solids, which contain cellulose, may then be sent toenzymatic hydrolysis with cellulase enzymes in order to convert thecellulose to glucose.

According to another example, the pretreated feedstock composition isfed to the fermentation without separation of the solids containedtherein. After the fermentation, the unhydrolyzed solids may besubjected to enzymatic hydrolysis with cellulase enzymes to convert thecellulose to glucose.

In at least one other example, the pretreated feedstock composition,together with any sugars resulting from hemicellulose hydrolysis, issubjected to cellulose hydrolysis with cellulase enzymes. Afterenzymatic hydrolysis, a major component of the resulting lignocellulosichydrolysate will be glucose, although pentose sugars derived from thehemicellulose component will be present as well.

Prior to hydrolysis with cellulase enzymes, the pH of the pretreatedfeedstock slurry is adjusted to a value that is amenable to thecellulase enzymes, which is typically between about 4 and about 6,although the pH can be higher if alkalophilic cellulases are used.

The enzymatic hydrolysis can be carried out with any type of cellulaseenzymes capable of hydrolyzing the cellulose to glucose, regardless oftheir source. Among the most widely studied, characterized, andcommercially produced cellulases are those obtained from fungi, such asTrichoderma spp., Aspergillus spp., Hypocrea spp., Humicola spp.,Neurospora spp., Orpinomyces spp., Gibberella spp., Emericella spp.,Chaetomium spp., Chrysosporium spp., Fusarium spp., Penicillium spp.,Magnaporthe spp., Phanerochaete spp., Trametes spp., Lentinula edodes,Gleophyllum trabeiu, Ophiostoma piliferum, Corpinus cinereus, Geomycespannorum, Cryptococcus laurentii, Aureobasidium pullulans, Amorphothecaresinae, Leucosporidium scotti, Cunninghamella elegans, Thermomyceslanuginosus, Myceliopthora thermophila, and Sporotrichum thermophile,and those obtained from bacteria of the genera Bacillus, Thermomyces,Clostridium, Streptomyces or Thermobifida. The cellulases typicallycomprise one or more CBHs, EGs, and β-glucosidase enzymes, and mayadditionally contain hemicellulases, esterases, and swollenins.

Following cellulose hydrolysis of the pretreated feedstock slurry, anyinsoluble solids, including but not limited to lignin, present in theresulting lignocellulosic hydrolysate may be removed using conventionalsolid-liquid separation techniques prior to any further processing.These solids may be burned to provide energy for the entire process.

It is also considered within the scope of the invention to produce thelignocellulosic hydrolysate by hydrolyzing the lignocellulosic feedstockin a single step with acid or alkali. This employs harsher conditions toeffect hydrolysis of both the hemicellulose and cellulose components ofthe feedstock (see, for example, U.S. Pat. No. 5,562,777, whichdescribes acid hydrolysis of cellulose and hemicellulose, the content ofwhich is hereby incorporated by reference herein). Furthermore, atwo-stage acid or alkali hydrolysis is also included within the scope ofthe invention.

Furthermore, it will be understood that, prior to fermentation, thelignocellulosic hydrolysate may be subjected to additional processingsteps. In at least one example, at least a portion of the mineral acidand/or organics acids, including acetic acid, present in thehemicellulose hydrolysate are removed from the lignocellulosichydrolysate, for example, by anion exchange (see, for example,International Patent Pub. No. WO 2008/019468, to Wahnon et al., which isincorporated herein by reference). Other processing steps that may beconducted prior to the fermentation include concentration by evaporationand/or reverse osmosis.

Components Present in the Lignocellulosic Hydrolysate

As discussed previously, hydrolysis of the hemicellulose and cellulosecomponents of a lignocellulosic feedstock yields a lignocellulosichydrolysate comprising xylose and glucose. Other sugars typicallypresent include galactose, mannose, arabinose, fucose, rhamnose, or acombination thereof. Regardless of the means of hydrolyzing thelignocellulosic feedstock (e.g., full acid hydrolysis or chemicalpretreatment with or without subsequent enzymatic hydrolysis), thexylose and glucose generally make up a large component of the sugarspresent in the lignocellulosic hydrolysate.

If the lignocellulosic hydrolysate is a hemicellulose hydrolysateresulting from acid pretreatment, xylose will be the predominant sugarand lesser amounts of glucose will be present, because a modest amountof cellulose hydrolysis typically occurs during pretreatment. Accordingto this embodiment of the invention, the xylose can make up betweenabout 50 and 90 wt % of the total carbohydrate content of thelignocellulosic hydrolysate. According to another embodiment of theinvention, the xylose makes up greater than about 30 wt % of the totalcarbohydrate content, between about 50 and about 90 wt %, or betweenabout 65 and about 90 wt % of the total carbohydrate content. Forexample, the xylose may make up greater than 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90 or 95 wt % of the total carbohydrate content. Itwill be appreciated by those of skill in the art that the relativeamount of xylose present in the lignocellulosic hydrolysate will dependon the feedstock and the pretreatment that is employed.

If the lignocellulosic hydrolysate results from hydrolysis of thecellulose and hemicellulose components of the feedstock, e.g., full acidor alkali hydrolysis, it will contain all of the sugars listed above,but will contain higher levels of glucose derived from the more completehydrolysis of the cellulose.

In addition to the aforementioned sugars, lignocellulosic hydrolysatesderived from lignocellulosic feedstocks contain a number of compoundsthat may or may not be inhibitory to the yeast. For example, furanderivatives such as 2-furaldehyde (furfural) and5-hydroxymethyl-2-furaldehyde (HMF) are inhibitory compounds thatoriginate from the breakdown of the carbohydrate fraction, namely xyloseand glucose, respectively. These compounds can be degraded further bypretreatment or hydrolysis into organic acids including acetic acid, aswell as formic and levulinic acids, which are also inhibitory.Additional organic acids found in the lignocellulosic hydrolysate thatmay be inhibitory to yeast include galacturonic acid, lactic acid,glucuronic acid, 4-O-methyl-D-glucuronic acid, or a combination thereof.Inhibiting phenolic compounds are also produced by the degradation oflignin; these include vanillin, syringaldehyde, andhydroxybenzylaldehyde. In particular, vanillin and syringaldehyde areproduced via the degradation of syringyl propane units andguaiacylpropane units of lignin (Jonsson et al. (1998) Appl. Microbiol.Biotechnol. 49:691).

As discussed previously, acetic acid is a component of lignocellulosichydrolysates that is highly inhibitory to yeast. The acetate arises fromacetyl groups attached to xylan and lignin that are liberated as aceticacid and/or acetate by exposure to acid or other chemicals thathydrolyze the feedstock (Abbott et al. (2007) FEMS Yeast Res. 7:819-33;Hu et al. (2009) Bioresource Technology 100:4843-47; Taherzadeh et al.(1997) Chem. Eng. Sci. 52(15):2653-59). Acetic acid has a pK_(a) ofabout 4.75 (K_(a) of 1.78×10⁻⁵), so that at pH 4.0 about 14.8 mole % ofthe acid is present as acetate. Thus, the species present in thelignocellulosic hydrolysate will depend on the pH of the solution.Although it should be appreciated that the practice of the invention isnot limited by the pH of the lignocellulosic hydrolysate, thefermentation is typically conducted at a pH at which acetate is thedominant species in solution. Acetic acid may be present in thelignocellulosic hydrolysate at a concentration of between about 0.1 andabout 50 g/L, about 0.1 and about 20 g/L, about 0.5 and about 20 g/L, orabout 1.0 and about 15 g/L. The inhibitory compounds set forth above arerepresentative of the compounds present in a lignocellulosic hydrolysateproduced from a lignocellulosic feedstock. A more extensive list ofcompounds that are present after pretreatment is provided in Klinke etal. ((2004) Appl. Microbiol. Biotechnol. 66:10-26), the content of whichis incorporated herein by reference. It will be appreciated that thesubstances present depend on both the raw material and the pretreatmentthat is employed.

Process for Fermentation of Lignocellulosic Hydrolysate by ModifiedYeasts

For the purposes of the fermentation process defined herein,fermentation means the conversion of the sugars or other carbon sourcespresent in a lignocellulose hydrolysate by a microorganism, such as ayeast cell, to a fermentation product, and/or the utilization of suchsugars and/or other carbon sources for cell growth. A fermentationproduct is defined as an organic molecule produced by, e.g., themodified yeast from the sugars and/or other carbon-containing substancespresent in the lignocellulose hydrolysate, as described hereinabove. Forexample, the fermentation product may be an alcohol, such as ethanol orbutanol, or a sugar alcohol, such as xylitol.

Preferably, the fermentation process is performed at or near thetemperature and pH optima of the fermentation microorganism. A typicaltemperature range for the fermentation of xylose to ethanol usingSaccharomyces spp. is between about 25° C. to about 37° C. or anytemperature therebetween, for example, from 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37° C. or any temperature therebetween, although thetemperature may be higher if the yeast is naturally or geneticallymodified to be thermostable. For example, the temperature may be fromabout 25° C. to about 55° C., or any value therebetween. The pH of atypical fermentation employing Saccharomyces spp. is between about 3 andabout 6, or any pH therebetween, for example, a pH of 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, or any pH therebetween.

The initial concentration of the fermentation microorganism will dependon other factors, such as the activity of the fermentationmicroorganism, the desired fermentation time, the volume of the reactor,and other parameters. It will be appreciated that these parameters maybe adjusted as desired by one of skill in the art to achieve optimalfermentation conditions.

A person of ordinary skill in the art will appreciate that thefermentation of lignocellulose hydrolysate using the modified yeastdescribed herein can be performed under aerobic, microaerobic, oranaerobic culture conditions. The appropriate ranges for duration oftime for various steps in the fermentation process will also be familiarto one of skilled in the art.

The fermentation medium containing the lignocellulosic hydrolysate mayalso be supplemented with additional nutrients. For example, yeastextract, specific amino acids, phosphate, nitrogen sources, salts, traceelements, and vitamins may be added to the hydrolysate slurry to supportgrowth and optimize productivity of the microorganism.

The fermentation may be conducted in batch, continuous, or fed-batchmodes, with or without agitation. The fermentation may also be conductedunder chemostat conditions. Preferably, the fermentation reactors areagitated lightly with mixing. In a typical commercial-scale process, thefermentation may be conducted using a series of reactors.

The fermentation may be conducted so that the modified yeast cells areseparated from the fermentation and sent back to the fermentationreaction. This may involve continuously withdrawing fermentation brothfrom the fermentation reactor and separating the yeast from thissolution by known separation techniques to produce a yeast slurry.Examples of suitable separation techniques include, but are not limitedto, centrifugation, microfiltration, plate and frame filtration,crossflow filtration, pressure filtration, settling, vacuum filtration,and the like.

The yeast slurry then may be treated with an oxidant to destroymicrobial contaminants. The oxidant may be selected from ozone,chlorine, chlorine dioxide, hydrogen peroxide, and potassiumpermanganate. For example, the oxidant may be chlorine dioxide; thisoxidant destroys microbial cells via the oxidation of aromatic andsulfur-containing amino acids of the intracellular enzymes. Chlorinedioxide is particularly suitable as an oxidant as bacteria are moresusceptible to its effects than yeast because most bacterial enzymes arelocated just inside the cell membrane whereas most yeast enzymes residedeeper inside the cell structure. Methods for using oxidants to destroymicrobial contaminants in yeast cultures are described in, e.g., Changet al. (1997) Appl. Environ. Microbiol. 63:1-6; International PatentPub. Nos. WO 2007/097874; WO 2009/026706; WO 2007/149450; and U.S.Patent Pub. No. 2009/0061490.

The entire contents of all references, patents, and patent applicationscited throughout this application are hereby incorporated by referenceherein.

EXAMPLES Example 1 Evolutionary Engineering of a Parental Yeast Strainto Produce a Modified Yeast Strain with Enhanced Fermentation of Xylosein Lignocellulosic Hydrolysate Example 1.1 Feed Preparation

Hydrolysate from a dilute acid-pretreated lignocellulosic biomass,conducted as set forth in U.S. Pat. No. 4,461,648 (incorporated hereinby reference), was concentrated by evaporation. Hydrolysate was mixedwith pure sugar and nutrient components according to media recipe #1(Media #1) in Table 2 (displaying components of Media #s 1, 2, 4, 5, and6). Salts, trace elements, and yeast extract were added, on a per literbasis, to the chemostat feed. The trace solution was prepared accordingto Verduyn et al. (1992) Yeast 8(7):501-17, which is incorporated hereinby reference.

TABLE 2 Media Recipes Media Media Media Media Media Compound #1 #2 #4 #5#6 Yeast Extract (g/L) 5 10 10 — — Peptone (g/L) — 20 20 — — (NH₄)₂SO₄(g/L) 9 — — 5 — KH₂PO₄ (g/L) 2 — — 3 — MgSO₄•7H₂O (g/L) 2 — —   0.5 —CaCl₂•2H₂O (g/L)   0.4 — — — — Trace elements (mL/L) 2 — — 1 — Glucose(pure) (g/L) 0-10 60 12 60  — Glucose (lignocellulosic — — — — 60hydrolysate) (g/L) Xylose (pure) (g/L) 0-40 20 — — Xylose(lignocellulosic 0-22 — 36 — 30 hydrolysate) (g/L) Acetic acid (g/L) 0-3.2 —  5   4.5   4.5 pH 5   4.8   4.8   5.5   5.5

Preparation of media for propagation of cells used to evaluatefermentability performance is described in Table 2 (Media #2).

Preparation of pure sugar media for fermentability assays was preparedaccording to Media #4 in Table 2. Lignocellulosic hydrolysate forfermentability assays was prepared by dilute acid pretreatment followedby enzymatic hydrolysis (Media #6).

Example 1.2 Inoculum Propagation for Chemostat

A parental S. cerevisiae strain LNH-ST was prepared as described in U.S.Pat. No. 7,527,927 and contained several copies of the plasmid pLNH-ST(FIG. 15), containing the XR-, XDH- and XK-encoding genes integratedinto its genome.

The parental strain LNH-ST (10⁸ cells) was used to inoculate a 2 Lbaffled flask containing 1000 mL of media containing Media #2 from Table2. Cells were cultivated for 48 h in a shaker incubator at 30° C. and160 rpm. The entire contents of the flask were transferred asepticallyto a controlled 7.5 L New Brunswick Bioflow III bioreactor containing 3L of Media #1 with 10 g/L glucose and 0 g/L xylose. The culture wasincubated for 3 h at the aforementioned conditions and then fed at arate of 3 g glucose/h for 18 h. This initial propagation step provided asufficiently large cell culture for the evolutionary engineeringexperiment.

Example 1.3 Chemostat Operation

Using increasing amounts of xylose originating from lignocellulosichydrolysate, the chemostat was fed sugars corresponding to a dilutionrate of 0.01-0.02 h⁻¹. The bioreactor, with a working volume of 4 L, wasmaintained at 30° C., and the pH was maintained at pH 4.5 using 15% v/vNH₄OH. The reactor was stirred at 150 rpm and sparger-aerated between0-0.8 standard liters per minute (slpm). The chemostat conditions weremaintained for approximately 2700 h, at which point the lignocellulosichydrolysate content had increased from 0 g/L to 22 g/L xylose.

As a person of ordinary skill in the art will be aware, the term“dilution rate” refers to the ratio of the feed rate to the volume ofculture in the vessel. That is, in a continuous stirred-tank reactor(CSTR) configuration, the volume of the culture remains constant and, assuch, the feed rate remains constant as well.

A colony was isolated from the chemostat after approximately 2700 husing a 1.5% agar plate containing 10 g/L yeast extract, 20 g/L peptone,and 17 g/L xylose from lignocellulosic hydrolysate. The isolated colonywith the adaptation to the lignocellulosic hydrolysate was named Y108-1.

Example 2 Measurement of Differential Fermentation Performance ofEvolved Yeast Strain Compared to Parental Strain

Fermentability performance: After 48 h of flask propagation in Media #2(Table 2) at 30° C. and 150 rpm, the parental strain LNH-ST and theevolutionarily modified Y108-1 strain were centrifuged at 3000×g for 5min and used to inoculate 400 mL of either pure sugar media (Media #5)or lignocellulosic hydrolysate (Media #6) to ˜8.0 g/L final biomassanaerobic 500 mL bioreactors. Prior to inoculation, the media wassparged with pure CO₂ for 2 min to ensure anaerobicity. Cells wereallowed to ferment at 30° C., 150 rpm until sugar exhaustion. CO₂production was monitored for the course of the fermentation and sampleswere taken for dry cell weight and HPLC analysis.

The samples were analyzed for cell mass using dry cell weight (Rice etal. (1980) Am. Soc. Brew. Chem. J. 38:142-45). For the fermentabilityanalysis, samples were taken from the bioreactors using a 10 mL syringe.From each sample, 2 mL volumes were centrifuged and the supernatantdecanted and filtered through a 0.2 μm syringe filter. Each supernatantsample was diluted with 5 mM sulfuric acid. All dilutions were analyzedfor glucose, xylose, xylitol, glycerol, and ethanol content on theAgilent 1100 Series Refractive Index Detector HPLC, while acetic andlactic acid were analyzed concurrently using an Agilent 1200 SeriesVariable Wavelength Detector HPLC. The column used for separation wasthe Varian Metacarb 87H Organic Acid column, maintained at 50° C. with a5 mM sulfuric acid mobile phase at a flow rate of 0.6 mL/min. The unitwas equipped with the 1100 Series Auto-sampler and Pumping System andcontrolled with the Chemstation software.

Using equivalent cell concentrations, the xylose consumption rate inlignocellulosic hydrolysate (FIG. 1A) shows that the modified strain(Y108-1) was able to convert over 60% of initial xylose to ethanol in 10h, compared to the parental strain (LNH-ST), which required 20 h. Themodified strain was also able to consume all available sugars, whereasthe parental strain ceased fermentation with about 8 g/L residualxylose. These differences are not observed in pure sugar mediafermentations (FIG. 1B).

Biomass and acetic acid production in the lignocellulosic and pure sugarfermentations were not affected by evolutionary modification (FIGS. 1Aand 1B). However, xylitol production was minimized by the fermentationof lignocellulosic hydrolysate with the modified strain (Y108-1), whichmay be indicative of an optimization of the redox imbalance (Table 3).S. cerevisiae harbors three cytosolic aldehyde dehydrogenases, two ofwhich are NAD⁺-dependent (encoded by ALD2 and ALD3) (Navarro-Avino etal. (1999) Yeast 15(10A):829-42) and one of which is NADP⁺-dependent(encoded by ALD6) (Meaden et al. (1997) Yeast 13(14):1319-27). Modig etal. ((2002) Biochem. J. 363:769-76) demonstrated that purified aldehydedehydrogenase from baker's yeast can utilize furfural as a substrate ina conversion of furfural to a less inhibiting analogue. The presence offurfural in lignocellulosic hydrolysates may result in increasedgeneration of NADPH required for continued xylose reductase activity,perhaps by increasing flux through the remaining NADP⁺-dependentisoform, ALD6p, the overexpression of which has been reported to conferresistance to furfural and HMF (Petersson et al. (2006) Yeast 23:455-64).

TABLE 3 Comparison of the Physiological Parameters of Fermentation inPure Sugar Media and Lignocellulosic Hydrolysate with the ModifiedStrain (Y108-1) and the Parental Strain (LNH-ST) Pure Sugar MediaLignocellulosic Hydrolysate Y108-1 LNH-ST Y108-1 LNH-ST Yield to ethanolfrom 0.49 +/− 0.00  0.48 +/− 0.01  0.44 +/− 0.01 0.45 +/− 0.01 glucoseand xylose (g/g) Yield to ethanol from ND** ND** 0.44 +/− 0.01 0.45 +/−0.01 glucose (g/g) Glucose uptake: q_(G) (g 1.87 +/− 0.03^(%) 1.67 +/−0.08^(%)  1.55 +/− 0.01^(%)  1.48 +/− 0.01^(%) sugar/g cells/h) Yield toethanol from ND** ND** 0.44 +/− 0.01 0.44 +/− 0.01 xylose (g/g) Specificxylose uptake: 0.10 +/− 0.01^(#) 0.11 +/− 0.02^(#)  0.15 +/− 0.00^(##) 0.07 +/− 0.01^(##) q_(x) (g sugar/g cells/h) Yield to xylitol from0.018 +/− 0.002  0.030 +/− 0.018   0.021 +/− 0.001*  0.044 +/− 0.004*xylose (g/g) *Denotes statistical differences in the mean value oftriplicate determinations (Student's t-test, p < 0.05). It is expectedthat values between media may differ, whereas differences betweenstrains are limited to xylose fermentation performance inlignocellulosic hydrolysates. Yields are based on sugar consumed andethanol or xylitol produced. ^(#)Based on an average specific xyloseconsumption rate between 4 and 9 g/L residual xylose. ^(##)Based on anaverage specific xylose consumption rate between 8 and 17 g/L residualxylose. **ND Not determined-the rapid rate of the co-consumption ofglucose and xylose in the Pure Sugar Media prevents calculation ofseparate xylose and glucose consumption rates. ^(%)Based on initialrate.

Table 3 indicates the physiological parameters for glucose and xylosefermentation for the modified and parental yeast strains. Evolutionarymodification of the parental strain (LNH-ST) to produce the modifiedstrain (Y108-1) did not affect yield or uptake of glucose. However,xylose utilization and uptake rate were improved in the modified strainY108-1. The Y108-1 strain also demonstrated decreased loss of xylose toxylitol production.

Example 3 Comparative Genomic Hybridization Analysis of the Modified andParental Yeast Strains Example 3.1 DNA Isolation

DNA isolation for comparative genomic hybridization (CGH) arrays wasperformed with the Promega A1120 Wizard genomics DNA purification kit(Fisher Scientific, Nepean, Ontario, Canada) according to themanufacturer's instructions. Briefly, Y108-1 and LNH-ST were grown for48 h in 3 mL of YPD (Yeast extract Peptone Dextrose) broth (10 g/L yeastextract, 20 g/L peptone, and 20 g/L dextrose) at 30° C. on an orbitalwheel at maximum rotation. Cells from 1 mL of culture were pelleted bycentrifugation at 14,500 rpm for 2 min and then resuspended in 293 μL of50 mM EDTA. Cells were lysed by addition of 7.5 μL of 20 mg/mL lyticaseand 60 min of incubation at 37° C. After cooling to room temperature,the sample was centrifuged at 14,500 rpm for 2 min and the supernatantwas removed. Nuclei lysis and protein precipitation solutions (300 μLand 100 μL, respectively) were added to the pellet, mixed, and vortexedfor 20 sec. The sample was incubated on ice for 5 min and thencentrifuged at 14,500 rpm for 3 min. The supernatant was transferred toa sterile microcentrifuge tube containing 300 μL of room temperatureisopropanol and mixed by inversion. After centrifugation at 14,500 rpmfor 2 min, the supernatant was decanted and the tube drained on a cleanpaper towel. The DNA pellet was then washed with 300 μL of roomtemperature 70% ethanol. The sample was centrifuged at 14,500 rpm for 2min and the ethanol supernatant was aspirated. The tube was drained(clean paper towel) and allowed to air-dry for 10 min. To rehydrate theDNA, 40 μL of DNA rehydration solution was added along with 1.5 μL ofRNase solution. After mixing, the tube was incubated at 37° C. for 15min. To allow for complete rehydration, the DNA pellet was incubatedovernight at 4° C. and then stored at −20° C.

Example 3.2 Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) measures DNA copy numberdifferences between test and reference genomes. Roche NimbleGen Inc.offers whole-genome CGH array products that measure DNA copy numbergains and losses across entire genomes. Whole genome tiling arrays of385K format were used (catalog number B2436001-00-01, design 2007-05-08SCER WG CGH). Duplicate genomic DNA samples of both Y108-1 and LNH-ST(biological duplicates) were shipped on dry ice to the Roche NimbleGenInc. facility (Reykjavik, Iceland) for use in the CGH experiment andwere labeled twice with opposite fluorophores (technical replicate) tocontrol for dye-incorporation bias. The genomic samples were labeled andhybridized, and arrays were scanned according to NimbleGen's internalprotocols (see cgh_userguide_version 4_(—)0.pdf). Probe intensities werequantified and processed with Roche NimbleGen NimbleScan and SignalMapsoftware, respectively, by NimbleGen.

Data from the CGH experiments were analyzed further with BioDiscoveryNexus software (El Segunda, Calif.). Significant copy number changeswere determined. Briefly, log-ratio data were loaded into theBioDiscovery Nexus Copy Number program for each hybridization. Regionsof copy number change were identified by optimized segmentation andcalling using BioDiscovery's proprietary Rank Segmentation algorithm. Areport was generated that included: a summary plot indicating the numberof oligonucleotides within each gene or open reading frame (ORF) showingdifferential hybridization to the sample and reference genomes, thefrequency of differential hybridization for each oligonucleotide acrossthe replicate hybridizations, the Quality score per each labeled DNAsample, and a list of all genes and their annotations for whichcontiguous regions of oligonucleotides showed differential hybridizationbetween the reference and sample genomes.

From these data, the genes in which a region of greater than about 60%displayed differential hybridization were selected as those whosedeletion or amplification are involved in improving tolerance to, andfermentation performance of, lignocellulosic hydrolysate (Table 4; seealso, e.g., FIGS. 3-7). Other changes observed in the genome of themodified strain (Y108-1) as compared to the genome of the parentalstrain (LNH-ST) include decreases in copy numbers of chromosomes 1 and 6and increases in copy numbers of chromosomes 9, 10, 11, 14, and 15.

TABLE 4 Genome Changes Identified in Modified Yeast Relative to ParentalYeast Copy number Copy number Gene or ORF change Gene or ORF changeCDC19 high gain ECM21 loss XKS1 high gain ILS1 loss ADH1 high gainYBL100W-C loss tE(UUC)B gain PRP6 loss BIK1 gain ERV14 loss RNQ1 gaintE(UUC)G2 loss FUS1 gain PRM8 loss PDI1 gain MST27 loss YCL042W gainSSA2 loss GLK1 gain ALD2 loss YCR045C gain ALD3 loss IMG1 gain YBR201C-Aloss BUD23 gain ARE1 gain YCL073C gain VBA3 gain DDI2 gain SNO3 gainSNZ3 gain

Example 4 Monitoring of Gene Expression in Y108-1 vs. LNH-ST StrainsExample 4.1 Isolation of RNA from Yeast Biomass

Triplicate samples with approximately 10 g/L of biomass were collectedat time points of 13.5 h and 23 h from the fermentation cultures of themodified (Y108-1) and parental yeast strains described in Example 1.Biomass was collected by centrifugation at 14,000 rpm for 3 min at 4°C., washed in cold sterile water three times, and frozen in liquidnitrogen. Yeast cells were resuspended in 600 μL RLT extraction buffer(Qiagen, Mississauga, Ontario, Canada) supplemented with 10 μL ofβ-mercaptoethanol and lysed with glass beads using a FastPrep (MPBiomedicals, Solon, Ohio) tissue disruptor (40 sec at a speed of 5 m/s).Total RNA was extracted using the yeast procedure outlined in QiagenRNeasy mini kit (cat #74104). RNA was quantified on Nanodropspectrophotometer (Thermo Scientific) using a conversion OD260 nm=1.0representing a concentration of 40 μg/mL. RNA integrity was verified byrunning the samples on a 2100 BioAnalyzer (Agilent Technologies,Mississauga, Ontario, Canada).

Example 4.2 Global Expression Profiling

For comparison of the global gene expression profiles of the modified(Y108-1) and parental (LNH-ST) yeast strains fermenting lignocellulosehydrolysate, custom S. cerevisiae 8×15K 60 mer microarrays (AgilentTechnologies) were designed using the online software e-array (AgilentTechnologies) using 6649 predicted ORFs (orf_coding_all.20080606.fastadownloaded from the URL: downloads.yeastgenome.org) and designing up totwo oligomers per ORF. Two labeling reactions, one with Cy3 and one withCy5, of each of the triplicate RNA samples prepared as in Example 4.1were performed as follows: Agilent's “Two-Color Quick Amp Labeling”procedure (version 5.7) was followed for labeling 500 ng of total RNA.

A total of six hybridizations (three RNA samples×two labeling reactions)were performed for each of the time points using the RNA from theparental yeast strain (LNH-ST) as a reference, and the RNA from themodified yeast strain (Y108-1) as the test sample. Hybridizations wereperformed overnight at 65° C. in a Robbins Scientific Model 400 ovenwith an Agilent Hybridization Oven Rotator. Following hybridization,microarrays were washed using the standard wash procedure as describedin the Agilent protocol. Slides were read using a Genepix 4200amicroarray scanner (Molecular Devices) at 5 μM resolution.Feature-finding was performed with Genepix Pro 6.0 (Molecular Devices).The results file (.gpr) was imported into Acuity 4.0 (Molecular Devices,Sunnyvale, Calif.) and Lowess normalization was applied. Analysis wascontinued using Lowess M log ratio as the datatype. Technicalreplications were combined and one-way ANOVA performed.

The results are presented in FIGS. 12, 13, and 14. A total oftwenty-seven genes showed increased expression in the modified yeast(Y108-1) relative to the parental yeast (LNH-ST) when grown underidentical conditions in media containing lignocellulose hydrolysates:GAL1, GAL7, GAL10, GAL80, PGM1, HXT1, HXT2, HXT3, GAL2, HXK2, GRR1,ERG1, ERG8, ERG10, ERG11, ERG20, ERG25, ERG26, ERG27, HMG1, CYB5, ERG2,ERG3, ERG4, ERG5, ERG24, and ERG28. An additional fourteen genes showeddecreased expression in the modified yeast (Y108-1) relative to theparental yeast (LNH-ST) when grown under identical conditions in mediacontaining lignocellulose hydrolysates: GAL3, GAL4, GAL11, PGM2, HXT4,HXT5, HXT6, HXT7, HXT9, HXT11, HXT12, SNF3, RTG2, and REG1. The majorityof these genes encode proteins that participate in hexose transport(FIG. 13 and Table 1), galactose transport and metabolism (FIG. 12 andTable 1), or ergosterol biosynthesis (FIG. 14 and Table 1).

Example 4.3 qRT-PCR Analysis

For quantitative real-time PCR analysis of selected gene transcripts,single-strand cDNA was prepared using 10 μg of total RNA from eachsample. RNA mixed with 1.5 μL of 100 μM of poly(T)₂₀ primer (Invitrogen,Carlsbad, Calif.), 2 μL of 25 mM dNTP (each dNTP at 6.25 nM) and volumebrought to 25 μL with nuclease-free water (Invitrogen). The RNA mixturewas heated on a thermal cycler at 65° C. for 5 min and then cooled to 4°C. To this mixture, 8 μL of 5× first-strand buffer (Invitrogen), 4 μl,of 0.1 M DTT (Invitrogen) and 1 μL of RNaseIN (Promega) were added.Reactions were mixed by vortexing and incubated at 42° C. for 2 min.Following this step, 2 μL of SuperScriptII (Invitrogen) were added. Thesynthesis reaction was continued for 60 min for all samples and theenzyme was inactivated by heat treatment at 70° C. for 15 min.

The expression levels of the Pichia stipitis XR and XDH genes (XYL1 andXYL2, respectively), as well as the S. cerevisiae XKS, FUS1, YCL042W,YCL073C, and VBA3 genes were determined by qRT-PCR using a StratageneMX3000P thermal cycler (Agilent). Transcript levels were determinedusing the standard curve method. Standard curves were constructed forconstitutively expressed reference genes RDN18 and ACT1 and for each ofthe gene-specific amplicons using primers indicated in Table 5 (SEQ IDNOs:1-18); RDN18 was used as the reference standard for highly expressedXYL1, XYL2, and XKS1 genes, and ACT1 was used as the reference standardfor the low- to moderately expressed FUS1, YCL042W, YCL073C, and VBA3genes. For generation of the RDN18 and ACT1 standard curves, equalaliquots of all collected cDNA samples were pooled, diluted 1:10, 1:20,1:100, 1:200, and 1:1000 in water, and used for qRT-PCR. To determinethe relative transcript level of each gene, individual cDNA samples werediluted 1:50, and 2 μL aliquoted in duplicate into a 96-well PCRmicro-well plate containing 18 μL of SYBR Green Master Mix composed of:10 μL of Maxima SYBR Green/Rox qPCR 2× master mix (Fermentas,Burlington, Ontario, Canada), 1 μL of forward and reverse primersolution at 10 μM, and 7 μL of nuclease free water. The PCR reactionconsisted of the following steps: I) 1 cycle of 30 sec. at 25° C., 10min at 95° C.; II) 45 cycles of 30 sec at 95° C., 20 sec at 55° C., and30 sec at 95° C. Analysis of the data was performed as described in theStratagene MX3000P manual for converting the fractional cycle at whichexponential product is reliably detected (threshold cycle or Ct) totranscript level. Standard curves were plotted for each gene to measurethe transcript level. These values were normalized to the referencegenes ACT1 or RDN18. The normalized transcript level for each gene forthe modified yeast strain was divided by that found for the sametranscript in the parental strain. The final value is the ratio of thenormalized relative transcript level of the target gene in the modifiedstrain (Y108-1) to that in the parental strain (LNH-ST).

TABLE 5 Primers for qRT-PCR SEQ ID Primer name Primer sequence 1 ACT1FTGGTTTCTCTCTACCTCACGCCAT 2 ACT1R TCGAAGTCCAAGGCGACGTAACAT 3 RDN18FAACTCACCAGGTCCAGACACAATAAGG 4 RDN18R AAGGTCTCGTTCGTTATCGCAATTAAGC 5XKS1F TCCGCTGCGGGACTACCTAAATAA 6 XKS1R CCAAGGGCACATGAGTTTGGTGTT 7psXYL1F TCGAATTCGCTCAATCCCGTGGTA 8 psXYL1R TTGAGCTGGAGACTTACCGTGCTT 9psXYL2F TGTTGGTGTCCACGCCTCTAAGTT 10 psXYL2R TGTGAGTAGCAGCACCAATGTCCT 11FUS1F AGGCTAGCGTCCAATTAGGGAAGA 12 FUS1R ATCCATCGGTATGAGTGGCCAGAA 13YCL042WF AGTGCCCAACTCAGCTTCCGTAAA 14 YCL042WR TCTCAGTGGCTTTGTGTAAGTCGTCG15 YCL073CF TGCCATATGGACACAAACCATGCC 16 YCL073CRAACCACAGCATCTCTTTCGGGTGA 17 VBA3F CGCTCATGAGTGCACAGCTTCAAA 18 VBA3RAGCACACCACCAAGAGTTGTACCT

The results are presented in FIGS. 8, 10, and 11. All three of the genesencoding the recombinantly introduced xylose metabolic pathway (the P.stipitis XYL1 and XYL2 genes encoding the XR and XDH enzymes,respectively, and the S. cerevisiae XKS1 gene encoding the XK enzyme)show increased expression in the modified yeast strain relative to theparental yeast strain when cultured under identical conditions in mediacontaining lignocellulose hydrolysates (FIG. 8), which is consistentwith the increased copy number observed for the ADH1 and CDC19 promoterslinked to the XYL1 and XYL2 genes, respectively. Similarly, other genesthat showed either an increase (FUS1, YCL042W, YCL073C, VBA3, BIK1,RNQ1, PDI1, GLK1, IMG1, BUD23, ARE1, DDI2, SNO3) or decrease (ECM21,YBL100W-C, SSA2, ALD2, ALD3, YBR201C-A) in copy number also showedincreased or decreased expression, respectively (FIGS. 10 and 11).

Example 5 Determination of Xylose Reductase, Xylitol Dehydrogenase, andXylulose Kinase Enzyme Activities in Parental and Modified Yeast StrainsExample 5.1 Preparation of Yeast Cell Lysates

Yeast samples with approximately 10 g/L of biomass were taken from eachof triplicate AFM flasks at time points of 13.5 h and 23 h intofermentation. Biomass was collected by centrifugation at 14,000 rpm for3 min, washed in cold sterile water three times, and frozen in liquidnitrogen. From these triplicate archive samples, a pooled mass of 0.0625g of cells was isolated and centrifuged for 10 min at 4150 RPM and 0° C.The pellet was then washed in ice-cold freeze buffer (10 mM potassiumphosphate buffer (KPB)+2 mM EDTA, pH 7.5), centrifuged again (4150 RPM,0° C.), resuspended in 4 mL of freeze buffer, and stored at −20° C.prior to preparation of cell-free lysates. Lysates were prepared fromthe frozen cells by thawing the samples, centrifuging for 3 min (4150RPM, 0° C.), and washing the pellet in ice-cold sonication buffer (100mM KPB+2 mM MgCl₂, pH 7.5); this was followed by another centrifugationand resuspension in 4 mL ice-cold sonication buffer with 40 μL1,4-dithiothreitol. The cells were then lysed by placing 0.5 mL of thecell suspension into a 2 mL Eppendorf tube containing 0.5 g ofacid-washed glass beads and agitated in an MP FastPrep-24 (MPBiomedicals) at 6 m/s for 50 seconds, followed by a cooling period (5min) on ice. The agitation/cooling process was performed twice. Lastly,the samples were spun down for 5 min at 14,500 RPM to remove celldebris, and the lysate was removed and kept on ice until use. Proteinconcentrations of the lysates were measured using the Lowry method(Lowry et al. (1951) J. Biol. Chem. 193:265-75) with bovine serumalbumin as a standard in a Cary300 spectrophotometer at 660 nm.

Example 5.2 Xylose Reductase (XR) Assay

The formation of D-xylitol (XOH) from D-xylose (X) was recorded bymonitoring the oxidation of NADPH and NADH (in separate reactions) at340 nm and 30° C. using the Cary300 Kinetics program. The reactionmixtures were performed in a 1 mL cuvette and consisted of DI water (800μL-X μL CFE), 50 mM Tris-HCl buffer (pH 7.0), 0.3 mM NADPH or NADH, andlysate. The cuvette was placed in the Cary300 and the background signalwas recorded. The reaction was started by adding the substrate (150 mMD-xylose) and terminated by simply removing the cuvette. Enzyme activitywas calculated from the change in absorbance difference between theslope of the background signal and the slope of the signal aftersubstrate addition per unit time using Beer's Law and the molarextinction coefficient of NAD(P)H (6.3 mL/mM). This value was thennormalized for the protein concentration in the lysate and finalactivity was expressed as U/mg protein.

The results are shown in FIG. 9A. The modified yeast strain (Y108-1)exhibits higher levels of xylose reductase activity, using either NADPHor NADH as a cofactor, than the parental strain (LNH-ST), consistentwith the increased copy number and expression of the P. stipitis XYL1gene.

Example 5.3 Xylose Dehydrogenase (XDH) Assay

The formation of D-xylulose from D-xylitol was determined by monitoringthe reduction of NAD+. The procedure for measuring XDH activity was thesame as previously described for xylose reductase. The reaction mixtureconsisted of deionized water (795 μL-X μL lysate), 100 mM glycine (pH9.0), 50 mM MgCl₂.6H₂O, 3.0 mM NAD+ and X μL lysate; the reaction wasinitiated by adding 300 mM xylitol.

The results are shown in FIG. 9B. The modified yeast strain (Y108-1)exhibits higher levels of xylitol dehydrogenase activity than theparental strain (LNH-ST), consistent with the increased copy number andexpression of the P. stipitis XYL2 gene.

Example 5.4 Xylulose Kinase (XK) Assay

The XK activity is measured by monitoring lactate formation frompyruvate via lactate dehydrogenase (LDH)-facilitated oxidation of NADH.The pyruvate originates from the previous reaction involving thedephosphorylation of phosphoenolpyruvate (PEP) via pyruvate kinase (PK)using ADP, which in turn was produced from the phosphorylation ofD-xylulose by XK.

Data was obtained using the same procedure as for XR and XDH. Thereaction mixture was composed of DI water (470 μL-X μL lysate), 0.1 MTris-HCl (pH 7.0), 50 mM MgCl₂.6H₂O, 5 mM ATP, 0.6 mM PEP, 20 U PK, 20 ULDH, 0.3 mM NADH and X μL lysate. The reaction was started using 40 mMD-xylulose.

The results are shown in FIG. 9B. The modified yeast strain (Y108-1)exhibits higher levels of xylulose kinase activity than the parentalstrain (LNH-ST), consistent with the increased copy number andexpression of the XKS1 gene in the modified yeast.

Example 6 Phenotypic Complementation

Yeast from the knockout and overexpression collections (Open Biosystems,Huntsville, Ala.) were identified through the corresponding gene copynumber gains or copy number losses in CGH analysis (Example 3.2) andthrough corresponding up- and downregulation in the transcriptionalmicroarray analysis (Example 4.2). Genes that were lost or gained or up-or down-regulated in Y108-1 compared to LNH-ST are thought to contributeto a beneficial phenotype with respect to fermentation and growth inlignocellulosic hydrolysate.

In order to enable xylose metabolism and compare the effects of deletingor overexpressing the CGH identified genes, the yeast strains in Table 6were transformed with the pLNH-ST plasmid (FIG. 15) using the lithiumacetate method of Gietz et al. ((2002) Methods in Enzymology 350:87-96).Briefly, yeast strains were inoculated into 20 mL of 2×YPD (10 g/L yeastextract, 20 g/L peptone, 60 g/L dextrose) and grown overnight.Afterwards, the cell titer was determined using a hemocytometer, and2.5×10⁸ cells were added to a 50 mL flask with 2×YPD. This mixture wasgrown until the cell titer was at least 2×10⁷ cells/mL (approximately 4hours of growth). The cells were harvested, washed in sterile water, andresuspended in 1 mL of sterile water. 100 μL samples were pipetted into1.5 mL microfuge tubes (one tube per transformation), centrifuged andthe supernatant removed. 360 μL of transformation mix (consisting of 240μL polyethylene glycol (50% w/v), 36 μL 1.0 M lithium acetate, 50 μLboiled single strand carrier DNA, and 34 μL purified pLNH plasmid) wasadded to the cells and vortexed. The cells were incubated in a 42° C.water bath for 90 minutes. After incubation, the cells were centrifuged,and the transformation mix was removed and resuspended in 1 mL sterilewater. Appropriate dilutions were performed and the cells were plated onSynthetic prop-out (SD)+xylose media consisting of 0.13% amino aciddrop-out media minus uracil, 0.17% yeast nitrogen base, 0.5% ammoniumsulfate and 2% xylose with 76 mg/L uracil added (parent and theknockouts) or without uracil (overexpressors) at pH 5.5. The plates forthe control and overexpressors contained 100 μg/mL geneticin, whereasthe knockout plates (already geneticin resistant) contained 800 μg/mLgeneticin. The cells were grown for approximately 4 days at 30° C.

To observe any differences in growth, the transformants were plated onglucose-free dilute lignocellulosic hydrolysate plates and compared togrowth on SD+xylose plates (FIGS. 16 and 17). Glucose was removed fromthe hydrolysate by fermentation with commercially available S.cerevisiae. The resulting fermentation broth was diluted to 20% of itsoriginal concentration (˜14 g/L xylose) and sterile filtered. This wasthen mixed with an autoclaved 30 g/L agar solution and allowed to cool,resulting in 10% lignocellulosic hydrolysate plates. Transformedcultures were incubated for approximately 5 days at 30° C.

TABLE 6 Yeast Strains Used in Phenotypic Complementation Study. Allstrains are derivatives of the parental strain BY4741 (MATa his3Δ1leu2Δ0 met15Δ0 ura3Δ0) (Open Biosystems Catalog No.) Overexpressed (OE)or Open Biosystems Catalog No. Target Gene Deleted (Knock out)YSC4515-98805369 BIK1 GST-Tagged Strain (OE) YSC4515-98805380 GLK1GST-Tagged Strain (OE) YSC4515-98805403 VBA3 GST-Tagged Strain (OE)YSC4515-98806294 SNO3 GST-Tagged Strain (OE) YSC4515-98806293 SNZ3GST-Tagged Strain (OE) YSC1021-551507 ALD2 Yeast Knock Out StrainYSC1021-551506 ALD3 Yeast Knock Out Strain YSC1021-552545 HXT5 YeastKnock Out Strain YSC1021-552203 HXT3 Yeast Knock Out StrainYSC4515-98807506 ERG3 GST-Tagged Strain (OE) YSC4515-98806621 ERG1GST-Tagged Strain (OE) YSC1021-555053 GAL1 Yeast Knock Out StrainYSC4515-98807524 GAL2 GST-Tagged Strain (OE) YSC4515-98805110 GAL10GST-Tagged Strain (OE) BY4741 Yeast Parental Strain

Example 7 Examination of Galactose Metabolism in a LignocellulosicBackground

LNH-ST and Y108-1 were grown in pure sugar media #5 (using 60 g/Lgalactose instead of glucose) at 30° C. for three days. To investigategalactose metabolism in a lignocellulosic background, sugar-depletedmedia was prepared. A fermentation was conducted with Y108-1 in order toconsume all available fermentable sugars. The fermentation beer was thensterile filtered, and to this 60 g/L galactose and 1 mL/L each ofvitamins and traces were added. LNH-ST and Y108-1 were inoculated intothe galactose-containing lignocellulosic hydrolysate and monitored forthree days. Sterile samples were withdrawn daily for HPLC analysis, asdescribed in Example 1. Under pure sugar conditions, yields and extentof galactose conversion were not significantly different. In alignocellulosic background, the parental strain LNH-ST fails to consumeall of the available galactose; whereas the modified strain Y108-1consumes all of the available galactose.

TABLE 7 LNH-ST and Y108-1 Were Grown in Either Pure Sugar Media orProcess Broth (Beer) Supplemented with 60 g/L Galactose Process Broth +Pure Sugar Galactose LNH-ST Y108-1 LNH-ST Y108-1 Y_(Ethanol/Gal) (g/g)0.34 0.36 0.33 0.35 Y_(Cells/Gal) (g/g) 0.10 0.09 0.07 0.10 % Galactoseconversion 100 100 92 100

What is claimed:
 1. A modified yeast strain capable of utilizing xylosein a lignocellulosic hydrolysate for growth or fermentation, comprisingan increase in copy number or expression of one or more genes selectedfrom the group consisting of ARE1, VBA3, RNQ1, FUS1, PDI1, BUD23, IMG1,DDI2, SNO3, SNZ3, YCL042W, YCL073C, YCR045C, and tE(UUC)B; a decrease incopy number or expression of one or more genes selected from the groupconsisting of ALD2, SSA2, MST27, PRM8, ERV14, ECM21, ILS1, PRP6,YBL100W-C, YBR201C-A, and tE(UUC)G2; or both (a) and (b), relative to aparental yeast strain from which the modified yeast strain is derived.2. The modified yeast strain of claim 1, further comprising an increasein copy number or expression of one or more genes selected from thegroup consisting of GAL1, GAL7, GAL10, GAL80, PGM1, HXT1, HXT2, HXT3,GAL2, HXK2, GRR1, ERG1, ERG8, ERG10, ERG11, ERG20, ERG25, ERG26, ERG27,HMG1, and CYB5; a decrease in copy number or expression of one or moregenes selected from the group consisting of GAL3, GAL4, GAL11, PGM2,HXT4, HXT5, HXT6, HXT7, HXT9, HXT11, HXT12, SNF3, RTG2, and REG1; orboth (a) and (b), relative to a parental yeast strain from which themodified yeast strain is derived.
 3. The modified yeast strain of claim1, further comprising (a) an increase in copy number or expression ofone or more genes selected from the group consisting of BIK1, GLK1,ERG2, ERG3, ERG4, ERG5, ERG24, and ERG28; (b) a decrease in copy numberor expression of ALD3; or (c) both (a) and (b), relative to a parentalyeast strain from which the modified yeast strain is derived.
 4. Themodified yeast strain of claim 1, wherein the modified yeast strain isalso capable of fermenting glucose in a lignocellulosic hydrolysate toethanol.
 5. The modified yeast strain of claim 1, wherein the modifiedyeast strain is also capable of fermenting arabinose in alignocellulosic hydrolysate to ethanol.
 6. The modified yeast strain ofclaim 1, wherein the strain is of the genus Saccharomyces.
 7. Themodified yeast strain of claim 6, wherein the parental yeast straincomprises introduced genes encoding xylose reductase, xylitoldehydrogenase, and xylulokinase.
 8. The modified yeast strain of claim7, wherein the modified yeast strain further comprises increases in copynumber or expression of the introduced genes encoding xylose reductase,xylitol dehydrogenase, and xylulokinase relative to the parental yeaststrain.
 9. The modified yeast strain of claim 6, wherein the parentalyeast strain comprises introduced genes encoding xylulokinase and one ormore xylose isomerase.
 10. The modified yeast strain of claim 1, whereinthe increase or decrease in copy number or expression of the one or moregenes is the result of an adaptive evolution technique.
 11. The modifiedyeast strain of claim 1, wherein the increase or decrease in copy numberor expression of the one or more genes is the result of geneticengineering.
 12. The modified yeast strain of claim 6, wherein theparental yeast strain is LNH-ST.
 13. The modified yeast strain of claim12, wherein the modified yeast strain capable of utilizing xylose isY108-1 (ATCC Deposit No. PTA-10567).
 14. A modified Saccharomycescerevisiae yeast strain capable of utilizing xylose in a lignocellulosichydrolysate for growth or fermentation, comprising (a) an increase incopy number or expression of one or more genes selected from the groupconsisting of ARE1, VBA3, RNQ1, FUS1, PDI1, BUD23, IMG1, DDI2, SNO3,SNZ3, YCL042W, YCL073C, YCR045C, and tE(UUC)B; (b) a decrease in copynumber or expression of one or more genes selected from the groupconsisting of ALD2, SSA2, MST27, PRM8, ERV14, ECM21, ILS1, PRP6,YBL100W-C, YBR201C-A, and tE(UUC)G2; (c) an increase in copy number orexpression of one or more genes selected from the group consisting ofGAL1, GAL7, GAL10, GAL80, PGM1, HXT1, HXT2, HXT3, GAL2, HXK2, GRR1,ERG1, ERG8, ERG10, ERG11, ERG20, ERG25, ERG26, ERG27, HMG1, and CYB5;and (d) a decrease in copy number or expression of one or more genesselected from the group consisting of GAL3, GAL4, GAL11, PGM2, HXT4,HXT5, HXT6, HXT7, HXT9, HXT11, HXT12, SNF3, RTG2, and REG1, relative toa parental yeast strain from which the modified yeast strain is derived,said modified yeast strain further comprising introduced genes encodingxylose reductase, xylitol dehydrogenase and xylulokinase.
 15. A methodof converting xylose in a lignocellulosic hydrolysate to a fermentationproduct, comprising the steps of: contacting the lignocellulosichydrolysate with the modified yeast strain according to any one ofclaims 1, 3, 4 or 14; and (b) recovering the fermentation product. 16.The process of claim 15, wherein the fermentation product is an alcoholor a sugar alcohol.
 17. The process of claim 16, wherein the alcohol isethanol and the sugar alcohol is xylitol.